MOAC MSc Miniproject Proposals Academic year: 2008 · Nicholas J Kruger and Antje von Schaewen (2003) The oxidative pentose phosphate pathway: structure and organisation. Current

MOAC

MSc Miniproject Proposals

Academic year: 2008

MOAC miniproject submissions 2008/9

Theory/computing projectsT1 van den Berg Kinetics of the transaldolase/transketolase system and its link to glucose metabolismT2 van den Berg Stochastic eradication processesT3 Hines Intelligent Systems approaches to protein secondary structure determination from UV circular dichroism and infra red dataT4 RodgerPM Passive transport of weak acids across lipid bilayers: insights from molecular dynamicsT5 Bretschneider Modelling diffusion of transmembrane cell adhesion moleculesT6 van den Berg Kinetics of the TCR/PMHCI/CD* complex and its role in regulation of immune specificityT7 van den Berg Predicting T cell receptor ligands using peptide libraies and Bayesian networksT8 Richardson Two-variable stochastic models of neuronal integrationT9 Richardson Measuring and analysing brain activity using EEGT10 Turner Membrane elasticity and energeticsT11 Vance Software facilitating the mapping of cis-regulatory module interactions in a genome wide mannerT12 (B4, PS11) Napier Time-resolved biosensor development. T13 Allen Parallel computer simulation of lipid bilayersT14 (PS13, B2) RodgerPM Structure/function studies on ALDC, part 1: Simulation studiesT15 Rigat Modelling of dose-response studies for novel organometallic anticancer complexes T16 Troisi Self-assembly of oligopeptides: a computational approach T17 Didelot Statistical analysis of adaptation in Salmonella T18 O'Connor Improving mass accuracy in FTICR mass spectrometry

Physical sciences projectsPS1 Macpherson Development of enzyme-based electrochemical biosensors for nitratePS2 Macpherson Isolation and Electrochemical Characterisation of Bacterial Nanowire Networks PS3 RodgerA DNA structures: what are they and how can they be probed by light or small molecules?PS4 RodgerA Peptidoglycan structure and reactionsPS5 RodgerA T cell antigen receptor membrane peptide: structure, function and activity mechanismPS6 Unwin Quantitative visualization of passive permeation across model cell membranesPS7 Unwin Development of electrochemical methods to facilitate real-time amperometric detection of catecholamine neurotransmitters in hippocampal brain slicesPS8 Dixon Structural Analysis of the Twin Arginine Translocase Pathway Protein TatA using Synthetic PeptidesPS9 Dixon Monitoring Membrane Protein Interactions using Fluorescence SpectroscopyPS10 (B3) Marsh Hormone binding analysis of a putative ABA receptor proteinPS11 (B4, T12) Napier Time-resolved biosensor development. PS12 (B11) Leszczyszyn Investigating the structural basis of metal binding and dynamics in metallothionein clusters PS13 (T14, B2) Wills Structure/function studies on ALDC, part 1: ChemistryPS14 Bugg Discovery and Characterisation of Lignin-degrading Bacteria using a Fluorescence Assay PS15 Bugg Labelling of Bacterial Cells with Fluorescent Peptidoglycan Intermediates PS16 Hicks Antibiotic peptides and their interactions with lipid membraPS17 Brown Development of Solid-State NMR as a Biophysical Probe PS18 Brown Solid-State NMR of a Membrane Protein

Wet biology projectsB1 Mitchell Structure-Function Analysis of the Carbohydrate Recognition Domain of the Human C-type Lectin DC-SIGNR Receptor Via Solution NMR.B2 (T14, PS13) Fulop Structure Structure/function studies on ALDC, part 2: CrystallographyB3 (PS10) Napier Hormone binding analysis of a putative ABA receptor proteinB4 (PS11, T12) Napier Time-resolved biosensor development. B5 Frigerio In vivo imaging of proteins that shape the plant ER B6 Easton Analysis of coupled translation in Mouse mRNAs B7 Freedman Using FRET to study the dynamics and biological function of PDIB8 Freedman Constructing mutations in the b’ and x regions of human PDIB9 Wall Exocytosis of adenosine in the brain?B10 Spanswick Perception of pain: the effects if upregulationof endocannabinoids on dorsal rot afferent-mediated synaptic transmissionB11 (PS12) Leszczyszyn Investigating the structural basis of metal binding and dynamics in metallothionein clusters B12 Spanswick The effects and mechansms of action of the gut-derived hormone oxyntomodulin on neuronal excitability of hypothalamic arcute nucleus neurones

B05

Project title: KINETICS OF THE TRANSALDOLASE/TRANSKETOLASE SYSTEM AND ITS LINKS TO GLUCOSE METABOLISM

Supervisor Name: Hugo van den Berg

Department: Mathematics Institute & MOAC ______________Building, Room: Coventry House 324 ___________

E-mail address: [email protected]______________ Phone number: 23698 ___________________

Project outline: A) GENERAL BACKGROUND In diabetes, the uptake of glucose into insulin-dependent cells is impaired, leading to a glucose shortage in these cells and elevated glucose levels in the blood plasma. These elevated glucose levels lead, in turn, to a surplus of glucose in non-insulin-dependent cells such as erythrocytes. In these cells, the functional compartments of hexose phosphates and triose phosphates contain metabolites at increased cytosolic concentrations, which leads to protein damage via “advanced glycosylated end products” which underlie much of the diabetes-related pathology. The pressure of elevated

hexose and triose phosphates can be relieved by rearranging these sugars into pentoses (2 hexoses and 1 triose yielding 3 pentoses) which can be cleared as ribose via the purine breakdown pathway. These sugar rearrangements are effected by two enzymes, transaldolase (TA) and transketolase (TK). Normally, these enzymes carry a flux from pentoses to hexoses/trioses as part of the pentose phosphate pathway (which generates

Project suitable as:

Project timing:(tick all that apply)

MOAC Mini Project Experimental biology x Slot 1 (26/3 to 16/05/08) Submission of poster: 8.59 am 19/05/08;

Talk at the annual conference

(8 weeks) Biophysical chemistry x Slot 2 (19/05 to 11/07/08) Submission of thesis: 8.59 am 14/07/08; viva to be scheduled in agreement with supervisor

x Mathematics/computing x Slot 3 (14/07 to 05/09/08) Submission of paper: 8.59 am 8/09/08 Talk: 3/10/08, MOAC Seminar Room

fruc-6P

glal-3P

erythrose-4P

sedoheptulose-7P

xylulose-5P

ribulose-5P

gluc-6P

fruc-1,6P2

Actn(OH)2P

glcnLactn-6P

glcnt-6P

CO2

[H]

[H]

ribose-5P

TK

TA

TPP

pentose phosphate pathway

glycolysis

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reducing equivalents for fatty acid synthesis), but in glucose-stressed cells, the net direction of the TA/TK-catalysed sugar rearrangement operates in reverse. Manipulation of the TA/TK system may have therapeutic potential: perhaps diabetic pathology can be averted by up-regulating these two enzymes. B) PROGRAMME OF WORK The flux through the TA/TK system depends on many factors: the concentrations of the two enzymes as well as of the metabolites fructose-6-phosphate, glyceraldehyde-3-phosphate, xylulose-5-phosphate, ribose-5-phosphate, erythrose-4-phosphate and sedoheptulose-7-phosphate. A mathematical model of the complicated enzyme kinetics has been developed. The miniproject can lead on to a multidisciplinary PhD project, with a co-supervisor in enzymology/proteomics. The project will involve the acquisition of experimental functional read-outs at the cellular level. The analysis of these data will be based on models of the sort developed in the miniproject. The aim of this miniproject is to investigate the cross talk between the normal regulation of glycolysis and the TA/TK system, to determine if artificial up-regulation of these enzymes will lead to the desired effect. In particular, the extrinsic (hormone-directed) regulation of the bifunctional enzyme is to be studied. To carry out this work, existing models of glycolysis and its regulation must be combined with the new model of TA/TK kinetics. C) SKILLS TO BE LEARNED The project involves literature study of core metabolism and its regulation (including existing models) and of relevant modelling methods, calculation of analytical results and computer simulations. D) RESOURCES REQUIRED None. E) STARTING REFERENCES Nicholas J Kruger and Antje von Schaewen (2003) The oxidative pentose phosphate pathway: structure and organisation Current Opinion in Plant Biology 6: 236-246 Shuichi Takayama, Glenn J. McGarvey, and Chi-Huey Wong (1997) MICROBIAL ALDOLASES AND TRANSKETOLASES:New Biocatalytic Approaches to Simple and Complex Sugars Annual Review of Microbiology 51: 285-310

Project title: STOCHASTIC ERADICATION PROCESSES




Project outline:

A) GENERAL BACKGROUND Stochastic extinction is an interesting phenomenon that occurs in disease eradication dynamics, which concerns the time-course of the elimination of proliferating diseased tissue (such as cancer cells or cells infected with an intracellular pathogen) by a given curative agent, either natural (immune cells) or artificial (drug). By definition, eradication requires that the number of diseased cells pass through small numbers. However, at low numbers the justification that underpins a deterministic treatment (e.g. in terms of ordinary differential equations) breaks down and stochastic effects become important. As a result, a “flare-up” of the disease may occur when the deterministic treatment predicts assured eradication; on the other hand, the deterministic treatment may show the disease to be ineradicable whereas extinction may in fact happen as a result of the stochastic bottleneck. These effects are important in the transition from acute to chronic viral infections and the treatment of cancer. It is of interest to obtain the probability of flare-up or eradication as a function of, say, diseased cell mass and dose of drug applied. The Figure shows a comparison between the fully stochastic approach (left) and the hybrid approach (right), where the x-variable is continuous-deterministic and the y-variable is discrete-stochastic. B) PROGRAMME OF WORK This project deals with a new way of looking at stochastic extinction processes. The conventional way out when the justification of the deterministic treatment fails is to analyze the underlying stochastic process (which was there “all







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along” of course, but which rarely receives explicit attention in deterministic accounts). Analysis of explicitly stochastic dynamics is cumbersome, even though there is a well-developed theory of such processes. However, we observe that determinacy only really breaks down for the number of diseased cells—the other components can still be treated as deterministic. This suggest a hybrid stochastic-deterministic system that is much more amenable to analysis than the full-blown stochastic system. The aim of this miniproject is to analyze various specific examples (equations and preliminary analysis are outlined in a preprint which can be obtained from Hugo), such as the eradication of cancer using an immunostimulatory agent and the elimination of a viral infection by the T cell response. C) SKILLS TO BE LEARNED The project involves a review of the relevant mathematical techniques, calculation of analytical results and computer simulations (code in the form of a Mathematica notebook is available).

MOAC mini-project proposal Submit up to TWO pages including this header page to http://www2.warwick.ac.uk/fac/sci/moac/intranet/miniproj/mp_proposal_submission_form_2008_9 by 9 February 2009. Any queries contact Mónica Lucena [email protected]. Delete all blue text before submission.

Project title: Intelligent Systems approaches to protein secondary structure determination from UV circular dichroism and infra red data

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Evor Hines (in collaboration with A. Rodger) ______

Department: Engineering ___________________________ Building, Room: A305 ____________________

E-mail address: [email protected] ______________ Phone number: 23246 ___________________

Supervisor’s advisor (for non-permanent members of staff or those on probation: academic who agrees to provide supervision

support to the supervisor and also agrees to meet the student briefly at least once a week): Name:__________________________________________

Department: ____________________________________ Building, Room: ________________________

E-mail address: __________________________________ Phone number: ________________________

Project outline:

A. Background to project Proteins are the key workhorse molecules in all cells. They only function when they are correctly folded at all levels: primary (sequence), secondary (local structural units), tertiary (folding of secondary structure) and even quaternary structure. For most applications it is important to know whether the protein is correctly folded in the environment in which it will be operating or from which it is delivered in the case of biopharmaceutical drugs. This means we need to be able to experimental measure and theoretically analyze protein structure data from aqueous solutions not only crystals. Two techniques widely used for protein secondary structure determination are circular dichroism (CD) and infra red absorbance. There are a number of algorithms that fit CD data from proteins of known CD and structure. There are also algorithms for using infra red data (though they are less extensively validated than the CD ones). There are to date no methodologies for combining the data from different techniques.

B. Programme of work

This project will involve assessing the CD and IR protein structure algorithms that are available and then developing one that can take data from both techniques (and others such as Raman and Raman Optical Activity) and combine them into a best fit structure. It is anticipated that Intelligent Systems techniques such as neural networks, genetic algorithms and fuzzy logic/systems type approaches will be adopted.

C. Skills to be learned

The project will require literature searching, understanding of spectroscopic data and its relationship to protein structure (to this end a limited amount of data collection may be undertaken), and appropriate data analysis techniques and Intelligent Systems.

D. Resources required (note total mini-project budget per student is £450 unless a special case is made)

** Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. However, all marks must be submitted at the latest by Sep 21 2009, 12 noon.

MOAC’s Annual conference is 27-30 May, 2009. Attendance is compulsory for students. They will present their first project during it. Supervisors are very welcome.

Project suitable as: (tick all that apply)

Project timing*:(tick all that apply)

MOAC Mini Project Experimental biology ✓ Slot 1 (19/3 to 15/05/09) Submission of poster by: 8.59 am 18/05/08; Talk at the annual conference

(8 weeks) Biophysical chemistry ✓ Slot 2 (18/05 to 10/07/08) Submission of thesis by: 8.59 am 13/07/08; viva to be scheduled in agreement with supervisor

✓ Mathematics/computing ✓ Slot 3 (13/07 to 04/09/08) Away for week 2ish, maybe another week too

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E. Outline of a literature review, including starting references Some examples of neural network papers may be found in: Protein Engineering vol.6 pp.383-390, 1993 ‘Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network’, M.A.Andrade, P.Chacón, JJ.Merelo1 and F.Morán. Many other references are collected in the web site Dichroweb (http://www.cryst.bbk.ac.uk/cdweb/html/home.html) run by Bonnie Wallace at Birkbeck.

MOAC mini-project proposalSubmit up to TWO pages including this header page to

Project title: Passive transport of weak acids across lipid bilayers: insights from molecular dynamics ____

Supervisor (the person who will be doing the day to day supervision of the mini-project):

Name: P.M. Rodger _______________________________

Department: Chemistry ____________________________ Building, Room: G107 ___________________

E-mail address: [email protected] ____________ Phone number: 23239___________________


support to the supervisor and also agrees to meet the student briefly at least once a week):

Name: __________________________________________

Department: _____________________________________ Building, Room: ________________________


Project outline:This project will use molecular dynamics to model the diffusion of small weak organic acids, such as acetic(CH3COOH) and propanoic (C2H5COOH) acids, across a lipid bilayer. Such simulations are important in providingmechanistic insight into the process, and thereby helping to develop better analytical models for passive transportof such molecules across membranes. Within this mini-project, coarse-grained molecular dynamics simulations willstudy the processes by which the weak acids enter, cross and then exit the membrane, and how this is affected bynature and packing of the lipids. Depending on progress, some targeted atomistic simulations may also beperformed. Methods for studying this process experimentally are the subject of a partner miniproject (P.R. Unwin).

Related Staff and Miniprojects: This miniproject is closely related to the experimental project “Quantitativevisualization of passive permeation across model cell membranes” supervised by P.R. Unwin. The scope of thesetwo miniprojects are such that they could readily be developed into a PhD project under the joint supervision ofProfs. Unwin (Chemistry), Allen (Physics) and Rodger.

Background:

There are a number of compounds that can enter cells without any active transport process occurring: they simplydiffuse along a concentration gradient. For example, protons can cross membranes more rapidly via the passivetransport of weak acids (protonophores) than is possible using “water wires” and membrane pores. Weak acidsalso form a class of drug whose activity depends on passive transport to specific intracellular sites [1]. Thetransport of weak acids across model membranes is therefore of considerable interest. [2]. A number ofphenomenological relationships exist that seek to predict the rate at which molecules will diffuse acrossmembranes, but all show significant quantitative discrepancies in the trends they predict with factors such asspreading pressure of the membrane, and size of the lipids that form the membrane. As such there is a need todevelop a better mechanistic understanding of the processes involved in passive transport, viz entry into themembrane across the head group region, diffusion across the hydrophobic region, and then exit across theopposite headgroup region. As each of these processes occurs on a molecular scale, they are amenable to studyusing molecular modelling methods.

Programme of Work:

Molecular dynamics simulations of a planar lipid bilayer in water will be performed using one of the existing coarse-grained potentials [3]. Initial simulations will focus on calculating the surface pressure-area isotherm for themembrane, so that system conditions for subsequent calculations can be chosen to correlate with those used inexperimental studies. Molecules of small, weak acids, will then be introduced into the simulations and used to studythe entry, intra-membrane diffusion and exit processes. Several different methods will be used for this purpose [4].Unconstrained dynamical simulations will used to determine diffusion coefficients and mechanisms for the intra-membrane transport. These simulations will also be analysed to determine whether such diffusion occurs by site-to-

Project suitable as: (tick all that apply) Project timing:(tick all that apply)

MOAC Mini Project Experimental biology Slot 1 (19/3 to 15/05/09)Submission of poster by: 8.59 am 18/05/08;Talk at the annual conference

(8 weeks) Biophysical chemistry Slot 2 (18/05 to 10/07/08)

Submission of thesis by: 8.59 am 13/07/08;viva to be scheduled in agreement withsupervisor

Maths/computing Slot 3 (13/07 to 04/09/08)Submission of paper by: 8.59 am 7/09/08Talk: 3/10/08, MOAC Seminar Room

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site hopping between special regions of the membrane. Entry and exit processes will be studied by imposing anexternal force on the weak acid, and using the linear response to determine entry/exit rates. Membrane surfaceswill also be analysed for spontaneous pore formation, and then acid molecules introduced into the vicinity of anysuch pores to determine whether these can substantially alter the entry/exit rate.

As this is an 8 week miniproject, it will be necessary to focus on one aspect of the possible variations in thissystem. Depending on progress, and the mutual interest student and supervisor, this focus may rest on variationsin the membrane properties as the lipid changes, differences in passive transport between several different acidpermeants, or on characterising different mechanisms for the different stages of transport.

Skills to be learned:

at a technical level, this project will develop expertise in running, verifying and analysing molecular dynamicsimulations, in both atomistic and coarse-grained forms, and into the protocols that are needed to use thesemethods for biomolecular simulations. On a more general level, the project will develop a fundamentalunderstanding of the properties of bilayer membranes on a molecular scale, and of how these properties affect thepassive transport of small molecules through membranes.

Resources Required: The project will require time on the University’s IBM cluster (Francesca). Access to asuitable linux workstation will be provided within PMR’s research lab. Other resources are standard computationalconsumables.

Literature:

[1] D. Cairns. Essentials of Pharmaceutical Chemistry. Pharmaceutical Press, 2003[2] T.-X. Xiang, B.D. Anderson, Biophysical Journal, 1998, 75, 2658[3] R. Faller, S.J. Marrink, Langmuir, 2004, 20, 7686[4] D. Bemporad, J.W. Essex, JW; C. Luttmann, J. Chem. Phys. B, 2004, 108, 4875

MOAC mini-project proposal

Project title: Modelling diffusion of transmembrane cell adhesion molecules__________________________


Name: Till Bretschneider_____________________________

Department: Systems Biology_________________________ Building, Room: Coventry House, #344________

E-mail address: [email protected]__________ Phone number: 024 76 1 50 252_____________



Name: Hugo van den Berg (yet to be confirmed)__________

Department: Systems Biology_________________________ Building, Room: Coventry House_____________

E-mail address: ___________________________________ Phone number: __________________________

Project outline:

Background to project

eCadherin is a trans-membrane molecule important in the regulation of cancer. eCadherin mediates cell:cell adhesion by binding the actin cytoskeleton within one cell and forming homo-dimers with eCadherin from neighboring cells. Recently, our collaborator Kurt Anderson (Beatson Institute of Cancer Research, Glasgow) has used photo-activation and -bleaching to study e-cadherin mobility in tumors in response to drug treatment. The immobile fraction value typically determined by FRAP (fluorescence recovery after photobleaching) appears to relate to the strength of adhesion between cells. But the half-time of recovery is much more difficult to interpret because of the many interactions which can influence this value. On the one hand, eCad can diffuse freely within the plasma membrane. However, eCad can also be endocytosed. Furthermore, eCad can transiently bind the actin cytoskeleton or eCad from neighboring cells. Binding and endocytosis influence the recovery rate in a manner which is difficult to predict from first principles. Therefore we would like to have a model of diffusion which allows us to vary these parameters in an effort to simulate the type of recovery data we obtain from activation/bleaching analyses. This would allow us to better understand on both short and long time scales how eCadherin dynamics are regulated by diffusion, binding, and endocytosis. The models we are going to build will be extensions of models we previously developed to analyze diffusion of membrane lipids in keratocytes (I. Weisswange, T. Bretschneider, K. Anderson. The leading edge is a lipid diffusion barrier. Journal of Cell Science, 118(19):4375-4380, 2005).

Programme of work

You will develop mathematical models and implement them in Matlab, which simulate various aspects of cell adhesion molecule membrane diffusion to help interpreting the dynamics we analyze using fluorescence microscopy.

Skills to be learnt

Development of reaction-diffusion type of models and their numerical approximation. Analysis of fluorescence microscopy data. Fitting models to data.

Resources required

£150 for a visit to meet our collaborator in Glasgow and a contribution to running costs of the image file server the student is going to use


MOAC Mini Project Experimental biology X Slot 1 (19/3 to 15/05/09)Submission of poster by: 8.59 am 18/05/08; Talk at the annual conference

(8 weeks) Biophysical chemistry X Slot 2 (18/05 to 10/07/08)Submission of thesis by: 8.59 am 13/07/08; viva to be scheduled in agreement with supervisor

X Mathematics/computing X Slot 3 (13/07 to 04/09/08)Submission of paper by: 8.59 am 7/09/08Talk: 3/10/08, MOAC Seminar Room

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Project title: KINETICS OF THE TCR/PMHCI/CD8 COMPLEX AND ITS ROLE IN REGULATION OF IMMUNE SPECIFICITY




Project outline: A) GENERAL BACKGROUND The cellular adaptive immune response is carried out by T cells, which constitute a major class of lymphocytes. These T cells specifically destroy those cells that have been infected by intracellular pathogens such as viruses; destruction of infected cells stems the spread of the pathogens, which need the intracellular environment in order to proliferate. This specificity of the T cell response depends critically on molecular recognition of the pathogen. It is mediated by the T cell antigen receptor (TCR) which interacts with antigenic peptides, bound by MHC molecules that are present on the surface of every cell. Since the system cannot anticipate the molecular identity of future pathogenic attacks, it maintains a large repertoire of TCRs, each of which has a distinct molecular specificity. At least one of these TCRs must interact well with a pathogenic peptide if the immune response is to be effective. This requirement means that each TCR must be able to interact with a large number of different peptides, since the repertoire, while vast, is not large enough to encompass a specific TCR for every possible peptide. The ability of the TCR to recognise multiple ligands is called TCR degeneracy. The immune system has various mechanisms that act to control this degeneracy. Moreover, chronic infection, autoimmune disease and cancer are all, in essence, failures to control TCR degeneracy. B) PROGRAMME OF WORK This project focuses on the regulation of TCR degeneracy by co-receptors. One such coreceptor is the CD8 molecule, which is found on T cells whose TCR







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interacts with the peptide/MHC class I (pMHCI) molecule. The co-receptor CD8 binds the TCR/pMHCI complex both at the TCR and at the MHCI. The TCR/pMHCI/CD8 contact has two effects: the TCR/pMHC bond is stabilized, and the signal transduction initiated by the TCR is accelerated. Both effects make the T cell more sensitive to pMHCI species (ligands) that bind the TCR with short half-lives, and less sensitive to ligands with longer half lives. Interestingly, co-receptor modulation of TCR degeneracy is dynamic: by up- or down-regulating its co-receptor, a T cell can ‘tune in’ to a given ligand.

The miniproject can lead on to a multidisciplinary PhD project, with an immunologist co-supervisor. Such a PhD project will involve the acquisition of experimental data on molecular rate constants as well as functional read-outs at the cellular level. The analysis of these data will be based on models of the sort developed in the miniproject. A very challenging question is whether it is possible to optimize the functional sensitivity of a given TCR clonotype for a ligand of interest by manipulating the interactions between the co-receptor CD8 and the TCR/pMHCI complex.

The aim of this miniproject is to analyse the kinetics of CD8 binding to the TCR and pMHCI. The mathematical tools are ordinary differential equations and Markov chains. The research objective is to characterize how the effects of CD8 at the molecular level translate to functional sensitivity at the cellular level, in dependence of the association and dissociation rates of CD8/TCR, CD8/pMHCI, and TCR/pMHCI. C) SKILLS TO BE LEARNED The project involves literature study of T cell immunity (including existing models) and of relevant modelling methods, calculation of analytical results and computer simulations. D) RESOURCES REQUIRED None. E) STARTING REFERENCES See references in preprint of a review of the subject, available from Hugo.

Project title: PREDICTING T CELL RECEPTOR LIGANDS USING PEPTIDE LIBRARIES & BAYESIAN NETWORKS




Project outline: A) GENERAL BACKGROUND The cellular adaptive immune response is carried out by T cells, which are a particular class of lymphocytes. These T cells specifically destroy cells that have been infected by intracellular pathogens such as viruses; this destruction of infected cells stems the spread of the pathogens, which need the intracellular environment in order to proliferate. The specificity of the T cell response depends on molecular recognition of the pathogen, mediated by the T cell antigen receptor (TCR), which interacts with antigenic peptides (epitopes) that are bound by MHC molecules, which are present on the surface of every cell. Since the system cannot anticipate the molecular identity of future pathogenic attacks, it maintains a large repertoire of TCRs, each of which has a distinct molecular specificity. At least one of these TCRs must interact well with a pathogenic peptide if the immune response is to be effective. This requirement means that each TCR must be able to interact with a large number of different peptides, since the repertoire, while vast, is not large enough to contain a specific TCR for every possible peptide. The ability of the TCR to recognise multiple ligands is known as TCR promiscuity or TCR degeneracy. The ability of the system to focus in on harmful cells while avoiding immunity directed against ‘self’ antigens, despite this degeneracy, is not yet fully understood. However, it has become clear in recent years that a plethora of regulatory mechanisms exists that dynamically modulate TCR degeneracy. Clinical manipulation of these mechanisms may lead to cure autoimmune disease and cancer. This requires quantitative insight into TCR degeneracy. For instance, clinically effective TCRs can be found more efficiently if one can accurately estimate the probability that a given peptide will be recognized by a given TCR.







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B) PROGRAMME OF WORK This project focuses on the peptide library method for the analysis of TCR degeneracy. In this method, the response of a particular TCR ‘clonotype’ against various mixtures of peptides, called libraries, is measured. Such a scan shows how likely it is that an agonist peptide (i.e. one that is well-recognized by this TCR) has amino acid residue X at position p. Here X ranges over the 20 proteogenic amino acids and p over the 9 positions in a nona-peptide, giving 180 data points. If the positions within the peptide were statistically independent with regard to TCR recognition, this data set already determines the probability that any given peptide is recognized by the TCR (up to a multiplicative constant). But such inter-positional dependencies do exist, in general.

The miniproject leads to a multidisciplinary PhD project, with a immunologist co-supervisor, in which the probe libraries are generated biochemically, and tested in bio-assay experiments involving several TCR clonotypes of clinical interest, generating graphical representations of the recognition pattern of these TCRs, with a view to validating these Bayesian network representations as epitope prediction tools and possibly develop them for pharma/therapeutical applications. Another possible research strand is to establish a correspondence between the Bayesian network and biophysical models of the TCR, relating the specificities to biochemical measures (affinity, on/off-rates, functional sensitivity), and accounting for shifts in recognition patterns for the same TCR when the co-receptor is up-regulated or down-regulated. The aim of this miniproject is to develop an iterative method to discover the most important positional dependencies. The idea is to design libraries that probe those inter-positional dependencies that are considered most important based on the present state of knowledge and to represent the data obtained from these probe libraries in a Bayesian network. The process is iterative: one starts with a trivial graph containing no positional interdependencies. At each stage, the graph can be used to calculate refined estimates of the recognition probability and to predict the next ‘most informative’ library probe. C) SKILLS TO BE LEARNED The project involves literature study of T cell immunity and of graphical inference methods, the development and implementation of the iterative algorithm and evaluation of the algorithm based on synthetic data sets derived from a known Bayesian network. D) RESOURCES REQUIRED None. E) STARTING REFERENCES See references in preprint of a review of the subject, available from Hugo.

MOAC mini-project proposal Project title: Two-variable stochastic models of neuronal integration

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Dr Magnus Richardson _____________________

Department: Warwick Systems Biology Centre _______ Building, Room: Coventry House 339 _______

E-mail address: [email protected] ___ Phone number: 50250 ___________________

Project outline: The computational power of the neocortex, the most recent part of the brain in evolutionary terms, arises from a combination of the rich response properties of neurons and the highly complex networks they form. Each cortical neuron can receive as many as 10,000 synapses. So, though the firing rate of individual neurons is low (around 0-5Hz) neurons are subject to a massive bombardment of synaptic current. This stochastic synaptic current can be reasonably modelled by a gaussian white-noise process with some leaky integration over the characteristic timescale of the membrane. This basic model has been studied since the 1970s. However, a variant, that includes an additional non-linear voltage term was recently shown by us (Badel et al, J. Neurophysiology 2008) to be an accurate model of biological neurons (see the figure - the agreement between model (green) and experiment (black) is the current state-of-the-art for reduced model fitting). The model that we used to fit to the data has a single variable for the voltage. Neurons, however, are geometrically extended cells that are composed of a cell body, the soma with long tree-like, branching dendritic structures attached. Synapses from other neurons form onto these dendrites in a well organised fashion, with certain classes of neuron making synapses near the cell body and others far out at the end of the dendrites. Because of this distribution of inputs along the dendrites, neurons are compartmentalised, in the sense that streams of information may be independently integrated at the dendrites or cell body. The net response of neurons is therefore highly complex due to the interaction of the stochastic synaptic streams with the spatially heterogeneous non-linear electrical response of the dendrites and cell body. The aim of this 8-week project will be to construct a two-variable mathematical model of neuronal integration corresponding to a coupled somatic and dendritic compartment. The project will require mathematical and programming skills and an interest in applying theoretical approaches to understand biological data. The student will receive training in MATLAB, stochastic calculus and data analysis. If you are interested in this project you are encouraged to contact me at the Systems Biology Centre to discuss the possibilities. References: Badel et al 2008. J Neurophys Richardson 2007 Phys. Rev. E (see my web site for PDFs) Resources required: £0



MOAC Mini Project Experimental biology X Slot 1 (19/3 to 15/05/09) Submission of poster by: 8.59 am 18/05/08; Talk at the annual conference

(8 weeks) Biophysical chemistry X Slot 2 (18/05 to 10/07/08) Submission of thesis by: 8.59 am 13/07/08; viva to be scheduled in agreement with supervisor

X Mathematics/computing X Slot 3 (13/07 to 04/09/08) Submission of paper by: 8.59 am 7/09/08 Talk: 3/10/08, MOAC Seminar Room

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EEG recordings showing 10Hz α-waves.


Project title: Measuring and analysing brain activity using EEG

Name: Dr Magnus Richardson _____________________

Department: Warwick Systems Biology Centre ________ Building, Room: 339 Coventry House _______

E-mail address: [email protected] ___ Phone number: 50250____________________

Second supervisor

Name: Dr Friederike Schlaghecken _________________

Department: Psychology __________________________ Building, Room: H248 ____________________

E-mail address: [email protected] ______ Phone number: 23178____________________

Background Electroencephalography (EEG) is a method for measuring brain activity non-invasively by using arrays of electric sensors placed over the scalp. The signal recorded at each electrode reflects the summed activity of hundreds of thousands of neurons and so extracting a useful signal that reflects brain activity is a considerable challenge. A standard solution to this problem is to present the task of interest repeatedly (from a few dozen to several hundreds of repetitions) and to average the EEG signals across all of these trials. Spontaneous, task-unrelated activity will be cleared from the signal by this procedure, whereas those signal modulations that are temporally linked to the task (event-related brain potentials or ERPs) will remain.

As well as the ERPs other signals, such as oscillations, are also present in EEG recordings. A strong frequency common to brain recordings is the α-rhythm at 10Hz, traditionally associated with the subject being in a relaxed state. Because of their large amplitude and high regularity, α-rhythms “pollute” the underlying ERP signal and so experimentalists often choose to discard data with these features. However, recent research has begun to highlight the possible role of these rhythms in cortical processes and suggests the possibility of extracting a useful signal from their strength, phase or coherence across the scalp.

While ERPs allow one to investigate localized, specific processes, large-scale oscillations like α-waves promise to provide an insight into general processing modes like top-down versus bottom-up processing, or controlled versus automatic processing.

Aims

The project aims to 1) analyse the large existing EEG data sets available at the Department of Psychology, and 2) generate predictions for future experiments to further our understanding of large-scale cortical oscillations. The student will help develop methods to analyse the properties of the α-waves measured across an array of scalp electrodes, and to examine whether their parameters correlate with the tasks undertaken by the experimental subject. The goal will be to test the hypothesis that α-waves do carry useful information. Depending on the progress and the interest of the student there will be the possibility also to participate in EEG recordings at the Department of Psychology.

A student with a good level of mathematical and programming skills would be well suited to the project. Training in MATLAB and basic data analysis techniques will be provided by Dr Magnus Richardson, and training in EEG recordings provided by Dr Friederike Schlaghecken. Consumables budget: £0




(8 weeks) Biophysical chemistry X Slot 2 (18/05 to 10/07/08) Submission of thesis by: 8.59 am 13/07/08; viva to be scheduled in agreement with supervisor

X Mathematics/computing X Slot 3 (13/07 to 04/09/08) Submission of paper by: 8.59 am 7/09/08 Talk: 3/10/08, MOAC Seminar Room

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MOAC mini-project: Membrane elasticity andenergetics

MST

February 9, 2009

Fluid membranes play an essential role in all living cells. They contain thecontents of cells and organelles but are much more than passive “sacks” - theymust permit the efficient, and highly selective transport, of cargo across themand also sense, e.g. their surface tension. Our theoretical understanding ofthese membranes is rapidly developing [1,2] but is still primitive in certaincases, particularly for membranes with many components. Experimentaldata is needed in order to determine the elastic and dynamic properties ofthese membranes and to constrain our theoretical models of their behaviour.Of particular interest would be experiments that measure the response tosteady, or time varying, forces applied to the membrane. The Atomic ForceMicroscope is a device that is perfectly suited to applying small, controlledforces at particular points on a membrane’s surface. Information on the force-displacement behaviour, and how this depends on frequency, can be used toinform theoretical models of the physics of “supported” fluid membranes, i.e.those that are coated onto a planar surface.The aim of this mini project will be to investigate the relationship betweendata of this sort and the underlying physical constants that parameterisethe membrane. The question is, “What can we hope to learn about fluidmembranes by conducting experiments of this sort”.

References

[1] http://www.warwick.ac.uk/ phscz/teach/leeds09/index.html[2] Statistical thermodynamics of surfaces, interfaces and membranes,S. A. Safran, Addison-Wesley.

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Matthew Turner Physics [email protected] Torturing fluid membranes

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MOAC mini-project proposal Submit up to TWO pages including this header page to http://www2.warwick.ac.uk/fac/sci/moac/intranet/miniproj/mp_proposal_submission_form_2008_9 by 9 February 2009. Any queries contact Mónica Lucena [email protected]. Project title: Software Facilitating the Mapping of Cis-Regulatory Module Interactions in a Genome Wide Manner

Supervisors (the person who will be doing the day to day supervision of the mini-project): Name: Keith Vance

Department: Systems Biology Centre _________________ Building, Room: BMRI, Room BM16 ____________

E-mail address: [email protected] _____________ Phone number: 02476 528380 _____________

Name: Sascha Ott

Department: Systems Biology Centre _________________ Building, Room: Coventry House, Room 327______

E-mail address: [email protected] _________________ Phone number: 02476 150258 _____________


support to the supervisor and also agrees to meet the student briefly at least once a week): Name: Georgy Koentges ___________________________

Department: Systems Biology Centre _________________ Building, Room: BMRI, Room M133 ________

E-mail address: [email protected] ____________ Phone number: 02476 574282 ____________

Project outline: Background The expression profile of a gene is controlled by genomic information processing devices called cis-regulatory modules (CRMs). A CRM is typically several hundred base pairs long and contains binding sites for multiple different positively and negatively acting transcription factors. We have used a novel bioinformatics tool based on billions of sequence alignments to attempt to identify CRMs in a genome wide manner. This has generated a dataset (CRM database) of putative CRMs containing approximately 35,000 conserved non-coding sequences of approximately 100-500 base pairs that lie within 100 kb of surrounding sequence for every annotated mouse Ensembl gene. CRMs are predicted to function to regulate gene expression by forming physical associations with each other and their target promoters. It is therefore proposed that the genome does not function in a linear manner but is organised into a three dimensional network of physical and functional interactions between different loci. This project aims to develop a set of software tools to design and analyse chromatin conformation capture (3C) based experiments to map the regulatory interactions of conserved CRMs in a high throughput manner. In these experiments interacting genomic elements are crosslinked together, purified, restriction enzyme digested, intramolecularly ligated and detected in a highly multiplexed ligation mediated amplification (LMA) reaction. This analysis will lead to the large scale functional annotation of the CRM dataset. Genomic regulatory interactions will be discovered in different experimental systems allowing the prioritisation of CRMs for further study.





MOAC Mini Project Experimental biology Slot 1 (19/3 to 15/05/09)


Mathematics/computing Slot 3 (13/07 to 04/09/08)

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Program of Work We have identified 117 genes encoding transcription factors and chromatin remodelling enzymes whose expression reproducibly changes during muscle differentiation. In this project we will generate BglII restriction enzyme digestion maps for up to 100 kb of genomic sequence surrounding these genes, depending on the distance to the nearest neighbouring gene. These restriction maps will be annotated onto the CRM database display of sequence conservation using Perl. We will write high level Perl scripts to call both BioPerl and bioinformatic Perl libraries to automatically design primers and analyse 5C experiments. The software will discover all BglII fragments containing conserved CRMs and design 20 nucleotide long oligos against either the top or bottom strand of the 3’ fragment ends. This will allow us to automatically design a library of unique ligation mediated amplification (LMA) primers to detect head to head ligation products between the BglII fragments containing the most highly conserved CRMs. In large scale 3C based experiments high throughput sequencing is used to detect ligation products representing genomic interactions. In this project we will create a database of all possible primer pair combinations that can be generated between the conserved CRM dataset. This will allow us to generate a quantitative map of the genomic regulatory interactions by matching each sequence read against this library using Sequence Search and Alignment by Hashing Algorithm (SSAHA). The results will be displayed as an interaction matrix with each box representing the number of times each primer pair was sequenced. Skills The candidate is expected to have a programming background and will be trained in the use of the bioinformatic software platform developed within the laboratory of Dr Sascha Ott. These software tools provide programmatic access to the CRM database of genome wide conserved regions and transcription factor binding site prediction data. This software platform is used for data mining in studies of transcriptional regulation in various organisms. Literature Review Tolhuis, B., Palstra, R. J., Splinter, E., Grosveld, F., and de Laat, W. (2002) Looping and interaction between hypersensitive sites in the active Β-globin locus. Mol Cell 10(6), 1453-1465 Dostie, J. et al (2006) Chromosome Conformation Capture Carbon Copy (5C): A massively parallel solution for mapping interactions between genomic elements. Genome Res 16(10), 1299-1309

MOAC mini-project proposal Submit up to TWO pages including this header page to http://www2.warwick.ac.uk/fac/sci/moac/intranet/miniproj/mp_proposal_submission_form_2008_9 by 9 February 2009. Any queries contact Mónica Lucena [email protected].

Project title: Parallel computer simulation of lipid bilayers

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Professor Michael P Allen

Department: Physics ______________________________ Building, Room: Physical Sciences, PS140 ___

E-mail address: [email protected] _____________ Phone number: ext 74415 ________________





Project outline:

A. Background to project. Particle-based simulation has been much used in recent years to obtain information about cell membranes, not only at the molecular level, but also to study much larger-scale phenomena. Very coarse-grained dissipative particle dynamics “DPD” models [1] have demonstrated lipid bilayer self assembly, a reasonable hierarchy of stable phases, and the effects of small molecules and transmembrane peptides on bilayer structure. By a suitable parameterization of the interactions [2] it has proved possible to investigate the mechanism of vesicle fusion [3]. Most of these studies focus on bilayers composed of a single lipid species, whereas real membranes consist of very many components. A starting point has been the study of two-component membranes [4] which addresses questions such as domain formation. More ambitious studies must combine the simplicity and efficiency of the DPD model with advanced Monte Carlo sampling techniques, such as multicanonical ensembles, and replica-exchange or parallel tempering. These approaches are readily parallelized, in the sense that a large number of simulations are run in parallel on a cluster computer, with regular exchanges of information or particle configurations. In effect, many state points are equilibrated together, for example spanning a wide range of temperatures, while allowing a given system to sample both low and high temperatures. The method may be systematically optimized by suitable choice of temperatures [5]. The project will build on a MOAC mini-project undertaken last year by Matt Bano. This studied the effects of transmembrane peptides on lipid bilayer structure, with the aim of simulating the formation of striated domains in the gel phase [6]. The project was successful, but two limiting factors were found: the intrinsically slow dynamics of the gel phase, and the strong effective forces between nearby peptides in the membrane. These problems can be attacked by temperature-driven parallel tempering, combined with umbrella sampling based on peptide-peptide distance [7].

The project is of strategic importance in transferring simulation expertise from the soft condensed matter group of MPA into the more biophysical application areas represented by MOAC.

B. Programme of work. Weeks 1-2. The student will adapt existing DPD simulation codes to run in parallel, exchanging information in a parallel tempering protocol. This is actually quite straightforward - it is an exercise in the undergraduate PX425 High Performance Computing course, for example - but will require learning the use of MPI (see below). Weeks 3-4. Lengthy runs on the HPC cluster, checking the proper equilibration of the lipid gel phase with higher-temperature, more mobile phases. Optimization of parameters such as the temperature distribution. Inclusion of DPD model peptides: singly and in pairs. Weeks 5-6. Incorporation of umbrella sampling based on peptide separation. This again is relatively straightforward. Further lengthy runs on the HPC cluster, studying the effects of the peptides on bilayer structure, and measuring the effective potential of mean force between peptides. Weeks 7-8. Possible simulations of many-peptide systems, if time permits. Preparation of materials for assessment.



Project suitable as: (tick all that apply) Project timing*:(tick all that apply)

MOAC Mini Project Experimental biology Slot 1 (19/3 to 15/05/09) Submission of poster by: 8.59 am 18/05/08; Talk at the annual conference

(8 weeks) Biophysical chemistry Slot 2 (18/05 to 10/07/08) Submission of thesis by: 8.59 am 13/07/08; viva to be scheduled in agreement with supervisor

Mathematics/computing Slot 3 (13/07 to 04/09/08) Submission of paper by: 8.59 am 7/09/08 Talk: 3/10/08, MOAC Seminar Room

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C. Skills to be learned. The student will learn parallel programming using MPI (Message Passing Interface), and will become experienced in the practical details of conducting large-scale simulations on a high-performance computer, including advanced aspects of statistical mechanics and simulation techniques, and the way to handle and analyse the data coming from such simulations. The simulation codes are basically ready-written, but prior experience of computer programming in Fortran or C is almost essential in order to understand them, as is familiarity with the contents of module CH926 “Computation II Molecular Modelling”.

D. Resources required None. Use of the CSC high-performance parallel computer is charged to departments, but this is supposed to be covered by fEC income from grants, and it is not really appropriate to charge MOAC projects.

E. Outline of Literature Review. The references given below will be the starting point for a literature review; [1] is a review of the relevant simulation field, [6] reviews the experimental situation, and the other papers give good examples of the various techniques in action.

References:

[1] M Venturoli, M M Sperotto, M Kranenburg, B Smit, “Mesoscopic models of biological membranes”, Physics Reports, 437, 1 (2006).

[2] LH Gao, J Shillcock, R Lipowsky, "Improved dissipative particle dynamics simulations of lipid bilayers", J Chem Phys, 126, 015101 (2007).

[3] LH Gao, R Lipowsky, J Shillcock, "Tension-induced vesicle fusion: pathways and pore dynamics", Soft Matter, 4, 1208 (2008).

[4] G Illya, R Lipowsky, JC Shillcock,"Two-component membrane material properties and domain formation from dissipative particle dynamics", J Chem Phys, 125, 114710 (2006).

[5] E Bittner, A Nußbaumer, W Janke, "Make Life Simple: Unleash the Full Power of the Parallel Tempering Algorithm", Phys Rev Lett, 101, 130603 (2008).

[6] B de Kruijff, JA Killian, DN Ganchev, HA Rinia, E Sparr, “Striated domains: self-organizing ordered assemblies of transmembrane a-helical peptides and lipids in bilayers”, Biol Chem, 387, 235 (2006).

[7] B West, FLH Brown, F Schmid, "Membrane-Protein Interactions in a Generic Coarse-Grained Model for Lipid Bilayers", Biophys. J. 96, 101 (2009).


Project title: Modelling of dose-response studies for novel organometallic anticancer complexes

Supervisor

Name: Fabio Rigat

Department: Statistics-WCAS Building, Room: Zeeman, D0.08

E-mail address: [email protected] Phone number: 50781

Supervisor’s advisor:

Name: Peter J Sadler

Department: Chemistry ____________________________Building, Room: C507 _______________________

E-mail address: [email protected] _____________Phone number: 23818/23653 _________________

Project outline:

We work on the discovery of new anticancer drugs based on transition metals such as ruthenium and osmium amongstothers. We have established structure-activity relationships and successfully investigated their mechanisms of action for severalyears. Ruthenium arene complexes, for example, have shown a high cytotoxic activity profile in a panel of human cancer cell lines.Their mechanism of action seems to involve DNA as the primary target site, showing preference for purine bases, in particularguanines. Emerging osmium compounds are promising drug candidates which also bind to DNA. The DNA targeting of thesecomplexes can be tuned to show a preference for guanine bases.

In addition to optimising drug design, we are fortunate to be able to carry out cell screening ourselves throughcollaboration with the department of Biological Sciences.Striving to overcome current chemotherapeutic drawbacks, including the emergence of drug resistance, we plan to designcytotoxicity assays involving combinations of drugs with different mechanisms of action using our cancer cell lines.

We plan to investigate how a small selection of our complexes combines with a set of reference agents including, forinstance, cisplatin, a widely-used DNA damaging anticancer agent. Specifically, we aim at establishing whether selectedcombinations of drugs individually acting via different mechanisms exhibit different cytotoxic effects when compared to theseagents alone. Does the order in which treatments are administered have any effect on the outcome? Do we observe eithersignificantly enhanced or reduced activity as a result of the combination?

We wish to create and validate dynamic models relating multiple dose levels and schedules to their observed levels ofcytotoxicity so as to explore rapidly possible synergistic or anti-synergistic effects of combinations of two or more drugs withdifferent mechanisms of anticancer action. The development of these models will include a phase of explorative data analysisusing non-parametric fitting of the observed relative cytotoxicity levels and non-parametric testing. These will provide overallquantitative measures of evidence against a null hypothesis of no difference among the cytotoxic effects of the chosen drugcombinations. A second phase of the analysis will implement statistical pharmacokinetic (PK) models, such as dynamic dose-response models, so as to provide a full probabilistic description of the relation between cytotoxicity and drug combinations.

The student will gain an appreciation of the relevance of the project, and the several factors affecting the statistical resultsthat the data will provide. To achieve this, the student will understand the way in which the cytotoxicity data are obtained, from thepreparation of the experiment, to every step of the protocol, to gaining the results and analysis of the data. Then, the student willprovide original contributions for the design and validation of the aforementioned statistical models for comparing the effects ofmultiple dose levels and schedules of a combination of at least two drugs. The project has the potential to contribute to new cancertherapies.

References

Bugarcic, T.; Habtemariam, A.; Stepankova, J.; Heringova, P.; Kasparkova, J.; Deeth, R. J.; Johnstone, R. D. L.; Prescimone, A.; Parkin, A.;Parsons, S.; Brabec, V.; Sadler, P. J. The Contrasting Chemistry and Cancer Cell Cytotoxicity of Bipyridine and BipyridinediolRuthenium (II) Arene Complexes. Inorganic Chemistry, 2008, 47, 11470.

Dougan, S. J.; Habtemariam, A.; McHale, S. E.; Parsons, S.; Sadler, P. J. Catalytic organometallic anticancer complexes. Proceedings ofthe National Academy of Sciences of the United States of America, 2008, 105, 11628.

Van Rijt, S. H.; Peacock, A. F. A.; Johnstone, R. D. L.; Parsons, S.; Sadler, P. J. Organometallic Osmium(II) Arene Anticancer ComplexesContaining Picolinate Derivatives. Inorganic Chemistry, 2009, 48, 1753.

Kostrhunova, H.; Florian, J.; Novakova, O.; Peacock, A. F. A.; Sadler, P. J.; Brabec, V. DNA Interactions of Monofunctional OrganometallicOsmium(II) Antitumor Complexes in Cell-Free Media. Journal of Medicinal Chemistry, 2008, 51, 3635.

Wakefield, J. The Bayesian Analysis of Population Pharmacokinetic Models. Journal of the American Statistical Association, 1996, 91, 62. Wakefield, J.; Bennett, J. The Bayesian Modeling of Covariates for Population Pharmacokinetic Models. Journal of the American

Statistical Association, 1996, 91, 917. Wakefield, J. An Expected Loss Approach to the Design of Dosage Regimens Via Sampling-Based Methods. The Statistician, 1994, 43,

13.

Project suitable as: Mathematics/computing Project timing: 19/3 to 15/05/09

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MOAC mini-project proposalSubmit up to TWO pages including this header page tohttp://www2.warwick.ac.uk/fac/sci/moac/intranet/miniproj/mp_proposal_submission_form_2008_9 by 9 February 2009. Any queries contactMónica Lucena [email protected].

Delete all blue text before submission.

Project title: Self-assembly of oligopeptides: a computational approach


Name: ALESSANDRO TROISI

Department: CHEMISTRY _________________________ Building, Room: CHEMISTRY (C511)

E-mail address: [email protected] _______________ Phone number: 02476523228



Name:__________________________________________


E-mail address: __________________________________ Phone number: _________________________

Project outline:

See next page

**Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course

Director. However, all marks must be submitted at the latest by Sep 21 2009, 12 noon.



MOAC Mini Project Experimental biology X Slot 1 (19/3 to 15/05/09)Submission of poster by: 8.59 am 18/05/08;Talk at the annual conference

(8 weeks) X Biophysical chemistr X Slot 2 (18/05 to 10/07/08)Submission of thesis by: 8.59 am 13/07/08;viva to be scheduled in agreement withsupervisor

X Mathematics/computing X Slot 3 (13/07 to 04/09/08)Submission of paper by: 8.59 am 7/09/08Talk: 3/10/08, MOAC Seminar Room

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Self-assembly of oligopeptides: a computational approach

MOAC miniproject proposed by Alessandro Troisi (Chemistry)

Background. Proteins under certain conditions aggregate toform fibers. Fibers formation is the main cause ofamyloidosis, a group of diseases including Alzheimer's orspongiform encephalopathies. Current research is focused onthe study of amyloid-like fibrils, formed by self-assembly ofoligopeptides and whose structure is known with atomic detail(Fig.1). The mechanism of fibers formation is poorlyunderstood as the formation of the fiber is a highlycooperative process which cannot be easily studied withstandard methods. The understanding of the molecularmechanism of aggregation may have impact on thedevelopment of a cure for amyloidosis.In our group we specialize in the simulation of molecular self-assembly using a variety of methods (Molecular Dynamics,Monte Carlo and Agent Based simulations). In particular weare interested in the development of ‘smart’ methods able to simulate the process of molecularself-assembly when it is highly cooperative, i.e. when it involves motions of portions of thesystem of different scales.

Program of work.1) Using an atomistic molecular mechanic program you will explore the potential energy

surface (i.e. the interaction energy) between two oligopeptides and will define anapproximate inter-molecular potential that best fits the atomistic computations.

2) Using either a cluster Monte Carlo algorithm or an Agent Based algorithm (developedin our group) you will study the formation of aggregates of the particles with theinteraction potential defined in part 1. The combination of 1) and 2), complementedwith some preliminary explorations carried out in our group, may form the basis for ascientific publication.

Continuation. As the experimental counterpart of this research is also carried out in Warwick(Dr. Pinheiro, Biology), a possible MOAC-PhD project can be envisioned.

Skills to be learned. (i) using Molecular Mechanics Packages, (ii) setting up advanced MonteCarlo simulations, (iii) additional programming skills and data analysis, (iv) working in aUnix environment and (v) using high performance computing.

Resources required. None (just stationery).

Further Readings. For a very short review see W.H.Binder and O.W.Smrza "Self-Assemblyof fibers and Fibrils" Angew.Chem.Int.Ed. 45 (2006) 7324. For a more detailed review ofseveral models of fibrillogenesis (unfortunately without a unitary framework) see E.Zerovnik"Amyloid-fibril formation - proposed mechanisms and relevance to conformational disease"Eur.J.Biochem 269 (2002) 3362. For a longer review including 3 theoretical models seeI.W.Hamley "Peptide Fibrillization" Angew.Chem.Int.Ed. 46 (2007) 8128. An importantexperimental paper (which could be used as a starting point for an in depth literature search isR.Nelson, et al., “Structure of the cross-β spine of amyloid-like fibrils”, Nature 435 (2005)773. For an idea on the advanced methods that we use in our group you can look at our ‘Anagent-based approach for modeling molecular self-organization’ Proc. Natl. Acad. Sci. USA102 (2005) 255 or ‘Self-assembly of sparsely distributed molecules: an efficient clusteralgorithm’ Chem. Phys. Lett. 458 (2008) 210. A visit to our lab to talk with current PGstudents can be easily arranged.

Fig.1:Oligopeptides form fibres withtheir -strands perpendicular to

the fibril axis


Project title: Statistical analysis of adaptation in Salmonella___________________________________


Name: Dr. Xavier Didelot ___________________________

Department: Department of Statistics _________________ Building, Room: Zeeman Building, Room D0.02

E-mail address: [email protected] _____________ Phone number: 02476 575754_____________



Name: Prof. Gareth Roberts _________________________

Department: Department of Statistics _________________ Building, Room: Zeeman Building, Room D0.04

E-mail address: [email protected]_______ Phone number: 02476 524631 ____________

Project outline:

Salmonella is a bacteria that causes grave diseases in humans resulting in around 300,000 deaths annually. Alarge collection of over 2000 isolates has been typed over the past five years using Multi-Locus Sequence Typing(MLST). MLST consists in sequencing 7 short fragments of genes for each isolate. The similarities and differencesobserved between the sequences reveal the relationships between the isolates. Approximately half of the typedisolates are from human sources and half from veterinary origins. It is clear from this data that certain types aremore likely to infect humans than animals, although the level of adaptation to the human host has never beenproperly quantified, and no evolutionary explanation has been given.

The aim of this project is to propose and apply a model for the evolution of adaptation in Salmonella. In particular,we would like to determine how often adaptation occurred during the evolution of the species, and to determinepoints where it happened to be used in future studies.

The first task will be to create (using existing methods) a phylogenetic tree showing how the isolates are related,and to look at how the isolates from human origin are distributed on this tree. We will then propose a stochasticmodel for how adaptation to the human host evolved in Salmonella. This model will finally be applied to the datausing a Monte-Carlo Markov Chain. Full training will be given on MCMC and programming techniques.

Starting references:

Maiden et al., 1998 Multilocus sequence typing: a portable approach to the identification of clones within populations ofpathogenic microorganisms. PNAS 95:3140-3145Torpdahl et al., 2005 Genotypic characterization of Salmonella by multilocus sequence typing, pulsed-field gel electrophoresisand amplified fragment length polymorphism Journal of Microbiological Methods 63:173-184








Mathematics/computing Slot 3 (13/07 to 04/09/08)Submission of paper by: 8.59 am 7/09/08Talk: 3/10/08, MOAC Seminar Room

http://www2.warwick.ac.uk/fac/sci/moac/intranet/miniproj/mp_proposal_submission_form_2008_9

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Project title: IMPROVING MASS ACCURACY IN FTICR MASS SPECTROMETRY

Supervisor

Name: Peter O’Connor

Department: Chemistry ______________________________

E-mail address: [email protected]_______________ Phone number: _____________________________

Background:

The general calibration equations for FTICR mass spectrometry are based around the following equation:

Where is the measured frequency and is the ideal cyclotron frequency . By Taylor

expansion of the square root terms, inversion, and rearrangement into an equation for m/z, one obtains this standard calibration equation:

Where A, B, and C are the calibration constants. In this equation, the "B" term is due to the magnetic field, and the "A" term is due to the electric field. B is typically a constant of ~108 in MKS units, A is ~104, and C (which should be very close to zero) takes other effects such as misalignment into account.

Clearly, the magnetic field is the primary determinant of ion oscillation frequency. However, these instruments routinely yield mass accuracy of 1 part in 106, or 1 ppm, so the other terms are critical. The "A" term is primarily due to the trapping voltage on the ICR cell, but coulombic repulsion among the ions trapped in the cell ("space charge") can contribute substantially resulting in tens to hundreds of ppm mass shifts. A simple trick has been used extensively to attempt to incorporate coulombic repulsion into the equation, and that is to substitute with , where D is another constant and I is the sum of total ion intensities in the spectrum. This method works fairly well, but fails in cases where there are low



MOAC Mini Project Experimental biology Slot 1 (26/3 to 16/05/08) Submission of poster: 8.59 am 19/05/08; Talk at the annual conference

(8 weeks) Biophysical chemistry Slot 2 (19/05 to 11/07/08) Submission of thesis: 8.59 am 14/07/08; viva to be scheduled in agreement with supervisor

x Mathematics/computing x Slot 3 (14/07 to 05/09/08) Submission of paper: 8.59 am 8/09/08

Talk: 3/10/08, MOAC Seminar Room

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intensity peaks which cannot be added into I, but which nevertheless affect the global space-charge environment in the cell.

A few years ago, it was recognized that the nonlinearity of this equation can be used to directly measure in the spectrum by measuring accurately a given mass difference at different points in the spectrum.

We propose, therefore, an iterative calibration method whereby, after initial assignment of the peaks in a mass spectrum, these proposed masses and mass differences are then used to automatically interpolate to calculate . If this works, the correction will be independent of I, and will include any low intensity contributions automatically.

Programme of work.

Determine if the parameter can be accurately calculated from mass differences in an assigned mass spectrum. If so, apply the method to a half dozen mass spectra and write a paper for submission to a mass spectrometry journal. Any necessary spectra will be provided by Prof. O'Connor, and if new spectra are needed, they will be acquired in Boston by his PhD students who remain there. A computer will need to be purchased for this work, but there's an already existing budget for this, and the computer will remain in the O'Connor group upon completion of the project.

Skills to be learned.

Calibration of FTICR mass spectra.

Literature work.

Matlab or other numerical analysis packages.

Writing a scientific publication

Resources required.

Computer time.

Mini-Lit review.

Zhang, L. K., D. Rempel, et al. (2005). "Accurate mass measurements by Fourier transform mass spectrometry." Mass Spectrometry Reviews 24(2): 286-309. Ledford, E. B., Jr., D. L. Rempel, et al. (1984). "Mass calibration law for quadrupolar potential." Anal. Chem. 56: 2744-2748. Bruce, J. E., G. A. Anderson, et al. (2000). "Obtaining more accurate Fourier transform ion cyclotron resonance mass measurements without internal standards using multiply charged ions." Journal of the American Society for Mass Spectrometry 11(5): 416-421. Easterling, M. L., T. H. Mize, et al. (1999). "Routine part-per-million mass accuracy for high-mass ions: Space-charge effects in MALDI FT-ICR." Analytical Chemistry 71(3): 624-632.


Development of enzyme-based electrochemical biosensors for nitrate


Name: Prof. Julie Macpherson

Department: Chemistry _________________ Building, Room: A106___________

E-mail address: [email protected] Phone number: 02476 573886____

This project is in conjunction with Prof. Martin Feelisch of the Warwick Medical Schoolbut will be largely based in Chemistry with Prof. Julie Macpherson.

Project outline:

Project outline:A: Project Background: Nitrate is a very common substance in the environment and its presence inbiological fluids, animal and plant tissues, as well as fresh and marine waters is of considerable interest,especially given that excessive ingestion of nitrate can have serious health implications and lead to e.g.,methemoglobinemia (blue baby syndrome) and gastrointestinal cancer. Consequently, maximalcontaminant levels of nitrate in drinking water and permissible nitrate load in salad, spinach and othervegetables are subject to strict regulation in the UK and elsewhere. Therefore, from an environmentalpoint-of-view, the ability to accurately determine nitrate levels in our water and food supplies is of vitalimportance. The recent appreciation that relatively low amounts of nitrite and nitrate exert beneficialbiological effects in their own right (1,2) and that nitrate is sequentially reduced to nitrite and further tonitric oxide not only in bacteria but also by mammalian cells (3,4) has also heightened the interest indetermining nitrate in food and water sources, and biological matrices.

The most common method for detecting nitrate is using spectrophotochemical means (such asfluorescence), where the chemically rather inert nitrate ion (NO3

-) must be reduced to the more reactivenitrite ion (NO2

-) before initiating the detection strategy. However, this technology is more suited tolaboratory-based measurements and is far from ideal for in-vivomeasurements, in-vitro cellular measurements (at the scale of individualcells) and on-line monitoring (e.g continuous monitoring in rivers andlakes). Thus measurement configurations are required that areamenable to scaling down in size and do not require de-gassing(removal of oxygen) from the solution. Electrochemistry represents anexciting approach to nitrate detection which is adaptable to both smallscale measurements and on-line monitoring. Importantly, the redoxactive enzyme nitrate reductase (NaR: see Figure 1), will exclusivelyinteract with nitrate via a two-electron oxidation process during whichnitrate is reduced to nitrite and the redox-active transition metal of theenzyme itself becomes oxidised. In the presence of an electron donormolecule (e.g. methyl viologen) the reduced form of the enzyme can be






(8 weeks) X Biophysical chemistry X Slot 2 (18/05 to 10/07/08)



Figure 1: Image of NaR

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regenerated for further use. The sensor detects the turnover of the electron donor molecule, by recordinga current, which is intimately related to the concentration of nitrate. In this way the NaR serves as abiocatalyst for nitrate detection, with the possibility of extended working lifetimes.

To produce an enzyme-based biosensor for nitrate the electrode must have two key components. Theunderlying electrode support material and the matrix, in which the enzyme and electron donor moleculeare co-located. For some applications it may be necessary to add an additional anti-fouling layer for usein complex fluids, such as in-vivo measurements. In this short project we will aim to develop anenzymatic platform for the electrochemical detection of nitrate. Carbon-based biocompatible supportmaterials will be used, such as conducting diamond,5 graphite or carbon nanotubes6 in combination withmethyl viologen as the electron donor molecule and polymeric-based, NaR impregnated, matrices, suchas Nafion or polypyrrole.

B: Programme of work: The overall aim of this project is to construct a working nitrate biosensor basedon the selectivity and redox-active properties of the NaR enzyme:

(i) In the first instance the aim will be to work with large-scale electrodes (mm size). A variety ofdifferent carbon electrodes will be tested in combination with various configurations ofenzyme loading; matrix identity and concentration of the electron donor molecule, in order toidentify the best conditions for a “workable” nitrate sensor. Key attributes of the sensor will bethe selectivity and sensitivity for nitrate; response time; lifetime; ease-of-use; ability to functionin complex media such as blood and tissues; and its sensitivity to interference by commonbiological constituents such as oxygen, glutathione, ascorbate etc. Some of theseexperiments will be carried out in direct comparison to a reference ion-chromatographicmethod established in Prof Feelisch’s laboratory.

(ii) If time allows methods to scale down the sensor to the micron range will be explored.

C: Skills to be learnt: The student will develop skills in the following areas:

(i) bioelectrochemistry (and the ability to work with redox-active enzymes);(ii) surface topographical characterization e.g. electron microscopy, atomic force microscopy;(iii) device fabrication;(iv) electrode testing in a variety of biologically relevant matrices and preparation of homogenates of

plant and animal tissues

D: Resources Required: (£450 total) – £400 for the purchase of the enzymes, electrode materials,device fabrication costs and consumables for HPLC and associated chemicals with biological matrices

E: References:

1. Lundberg JO, Weitzberg E, Gladwin MT.The nitrate-nitrite-nitric oxide pathway in physiology andtherapeutics. Nat. Rev. Drug Discov. 2008 Feb;7(2):156-67.

2. Butler AR, Feelisch M. Therapeutic uses of inorganic nitrite and nitrate: from the past to the future.Circulation. 2008 Apr 22;117(16):2151-9.

3. Jansson EA, Huang L, Malkey R, Govoni M, Nihlén C, Olsson A, Stensdotter M, Petersson J, HolmL, Weitzberg E, Lundberg JO. A mammalian functional nitrate reductase that regulates nitrite andnitric oxide homeostasis. Nat Chem Biol. 2008 Jul;4(7):411-7.

4. Cornelius J, Tran T, Turner N, Piazza A, Mills L, Slack R, Hauser S, Alexander JS, Grisham MB,Feelisch M, Rodriguez J. Isotope tracing enhancement of chemiluminescence assays for nitric oxideresearch. Biol Chem. 2008 Nov 29.

5. Hutton LA, Newton ME, Unwin PR and Macpherson JV, Amperometric Oxygen Sensor Based on aPlatinum Nanoparticle-Modified Polycrystalline Boron Doped Diamond Disk Electrode, Anal. Chem.2009 in press

6. Bertoncello, P, Edgeworth, JP, Macpherson JV and Unwin, PR Trace Level Cyclic VoltammetryFacilitated by Single Walled Carbon Nanotube Network Electrodes, J. Am. Chem. Soc. 2007, 129,10982-10983.


Project title: Isolation and Electrochemical Characterisation of Bacterial Nanowire Networks

Supervisors (the person who will be doing the day to day supervision of the mini-project):

Name: Prof. Julie Macpherson and Prof. Greg Challis __

Department: Chemistry_____________________________ Building, Room: A106/C519 _______________

E-mail address: [email protected] and [email protected]

Phone number: 73886 _____________________________

Project outline:

A: Background: Recently, it has been reported that avariety of bacteria, including dissimilatory metal reducingbacteria (such as Shewanella oneidensis MR-1andGeobacter sulfurreducens), oxygenic phototrophiccyanobacteria and fermentative bacteria, produceelectrically conductive pili that act as nanowires (Figure1), which are thought to be active as electron transfersites. If these pili can be harnessed this finding has majorpotential implications in diverse areas such as alternativeenergy production (e.g. microbial fuel cells), bacterialcell-to-cell communication and nanoelectronicapplications. To fully exploit the potential of thisremarkable finding, a fundamental understanding of theelectrochemical properties and molecular composition ofbacterial nanowires is required. The new field created bythe discovery of bacterial nanowires is evolving rapidly(as witnessed by 76 citations to the original Nature report in July 20051 and 30 citations to the secondreport in PNAS, published in July 20062) and is thus a fast moving research area. The project will bejointly supervised by Prof. Greg Challis (chemical biologist) and Prof. Julie Macpherson(electrochemist/surface scientist).

The specific objectives of this mini-project are:

Production of S. oneidensis bacteria with pili appendages (the purported ‘nanowire’ structures) High resolution structural characterisation of nanowire-producing bacterial cells Electrochemical investigation of the role of the pili in controlling the electrochemical transfer propertiesof a connected S. oneidensis network

B: Programme of work: (i) Bacterial nanowires will first be produced using S. oneidensis strain MR-1by culturing in continuous flow reactors (fermenters) operating in chemostat mode in a chemically-defined medium under oxygen-limiting conditions.2 This equipment was recently purchased via an award









Figure 1: SEM image of S. oneidensis cellsinterconnected by nanowire ‘pili’.

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from the Warwick Research and Development Fund and preliminary work has already shown that we canproduced bacteria using it. The required strain is readily available from the ATCC global bioresourcecentre.3 Bacterial nanowire production will take place in the laboratories of Prof. Challis. Once conditionshave been elucidated for immobilization of the S. oneidensis cells onto a solid support, structural highresolution characterisation of the anaerobically prepared cells will be carried out using state-of-the-artfield emission scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM). The formerinstrument (housed in Physics in the Microscopy suite) offers a larger field of view and will provideinformation on the long range ordering i.e. the distances over which the S. oneidensis cells are inter-connected by protruding pili. In contrast, AFM enables accurate height and length information to beelucidated, at the sub-nm level. Crucial to the project will be elucidation of the electron transfer andtransport properties of the S. oneidensis cell and the role of the purported ‘electrically conductive’ piliwhich are thought to act as nanowires.

(ii) Once immobilized, experiments will be conducted to elucidate the role of the nanowires incontrolling the conductivity of a network of interconnected S. oneidensis cells i.e. do the cells electricallycommunicate between each other using the pili; and the formal reduction potentials of the system werenot established. In order to exploit the potentially powerful properties of these biological nanowires theabove questions must be answered.

Using scanning electrochemical microscopy (SECM) techniques, unique to Warwick,4,5 and basedin the laboratories of Macpherson and Prof. Unwin, we will aim to resolve these issues in this shortproject. Networks of the cells will first be adhered onto insulating surfaces, in this way any informationextracted from conductivity comes from the S. oneidensis cellsthemselves and not the substrate. Working under physiological pHconditions, a small microelectrode placed close to a network of S.oneidensis cell can be used to electrogenerate an electron acceptormolecule (B) of defined redox potential, from solution species A trappedin the tip-substrate gap. B diffuses to the surface where it intercepts thebacterial network of cells. If B has the appropriate redox potential forelectrons to transfer from the network it is reconverted back to A, thusproducing an enhanced current at the tip electrode (compared with if thesurface was insulating), see Figure 2. Different factors can affect thedetected current magnitude: (i) the redox potential of species A/B. Byexploring a range of redox couples, with different electrochemicalpotentials, it will be possible to elucidate the potential threshold forelectron transfer; (ii) the size of the tip of the electrode. In these studies alarger sized tip (e.g. 25 m diameter) will be employed where the tip sitsabove many cells. For a tip of this size, the current due to feedback ofthe mediator from one single cell (not in electrical contact with its neighbors) will be negligible, howeverif the cells are electrically connected by the pili nanowires, a significant feedback current should beobserved. By recording tip approach curves with different mediator concentrations it will be possible toextract a value for the lateral conductivity of the network,5 should the cells be electrically connected.

C: Skills to be learnt: The student will gain valuable skills in bacterial cell production usingfermentation techniques, high resolution microscopy (FE-SEM and AFM) and electrochemicalcharacterization techniques, including SECM for elucidating the lateral conductivity of the bacterialnanowire network.

D: Resources required: £300 required for growth of bacteria in the fermenter, to include e.g. growthmedia, plastic ware, pipette tips etc

E: References:

1. G. Reguera, K. D. McCarthy, T. Mehta, J. S. Nicoll, M. T. Tuominen and D. R. Lovely, Nature, 2005, 435, 1098-1101.2. Y. A. Gorby et al., Proc. Nat. Acad. Sci, 2006, 103, 11358-113633. www. atcc.org4. M. A. Edwards, S. Martin, A. L. Whitworth, J. V. Macpherson and P. R. Unwin, Physiol. Meas. 2006, 27, R635. D. Mandler and P. R. Unwin J. Phys. Chem. B, 2003, 107, 407

Figure 2: SECM schematic ofnanowire regeneration of species A


Project title: DNA structures: what are they and how can they be probed by light or small molecules? ____

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Alison Rodger ______________________________

Department: Chemistry _____________________________ Building, Room: B607 ____________________

E-mail address: [email protected]_______________ Phone number: 07876218199/48199/74696/23234


support to the supervisor and also agrees to meet the student briefly at least once a week): Name: __________________________________________

Department: _____________________________________ Building, Room: _________________________


Project outline:

A. Background to project The structure of DNA plays a key role in its function in biological systems. However, our knowledge of what structures are adopted when and why is remarkably limited. Methods for identifying when a given structure is found are limited and often not suited to probing DNA in a dynamics situation. We have recently found that by measuring the linear dichroism spectrum of DNAs to much lower wavelength than had been possible previously that the spectrum changes significantly as a function of sequence and salt concentration in a manner consistent with us having found a spectral signature for single stranded DNA. In other cases molecules have been found to be selective for single stranded, to double stranded, or triplex, 1 or quadruplex, 2 or for special junctions such as the three-way junction induced by a di-iron triple helicate molecule.3

B. Programme of work This project will involve

1. Surveying the methods currently used to identify DNA structures in solution. Determine sequence preferences for different structures. This will focus on DNA curvature, plasmid structures adopted, and literature estimates of single stranded (ss) percentages in double stranded DNA.

2. Measuring the linear dichroism 4 of DNA as a function of sequence, temperature, salt and pH to investigate its utility as a measure of dynamic regions of single stranded DNA within duplex structures. This will focus on AT rich DNAs in order to create maximal ss regions. It will also involve investigating percentages of ss DNA using ss-binding dyes. DNAs will be at least 1000 bp in length ranging up to ~10,000 bp.

3. Screening a range of different cationic molecules (including those designed and synthesized by Prof. Peter Scott and colleagues in the department of chemistry) as selective probes of different DNA structures using a range of spectroscopic techniques including LD, CD, DLS, absorbance






(8 weeks) ✓ Biophysical chemistry ✓ Slot 2 (18/05 to 10/07/08) Submission of thesis by: 8.59 am 13/07/08; viva to be scheduled in agreement with supervisor

Mathematics/computing ✓ Slot 3 (13/07 to 04/09/08) Away for week 2ish, maybe another week too

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and fluorescence. The actual details of this part of the project will depend on results from 1 and 2 and the students own preference. The student will be responsible for designing this part of the project in collaboration with AR and PS.

4. More extensive studies of a selected class of DNA structures and its role in biology. This part of the project will probably be an alternative to 3. and will also be designed by the student depending on their own interests.

C. Skills to be learned The project will require laboratory sample handling of nucleic acids, use of a range of spectroscopic and related techniques including LD, CD, DLS, absorbance and fluorescence. Analysis of spectroscopic data to give steady state structures and binding constants. The project will also involve surveying the DNA structure literature and data bases.


A range of DNAs will be required for the project at estimated cost of £250–£300.

E. Outline of a literature review, including starting references (1) Kim, S. K.; Sun, J.-S.; Garestier, T.; Hélène, C.; Nguyen, C. H.; Bisagni, E.; Rodger, A.; Nordén, B. “Binding geometries and protonation states of triple helix selective benzopyrido[4,3-b]indole ligands complexed with double and triple helical polynucleotides” Biopolymers, 1997, 42, 101– (2) Đapic, V., Abdomerovic, V., Marrington, R., Peberdy, J.C., Rodger, A., Trent, J.O., Bates, P.J., “Biophysical and biological properties of G-quartet forming oligonucleotides” Nucleic Acids Research 2003, 31, 2097−2107 (3) Oleksi, A.; Blanco, A.G.; Boer, R.; Usón, I.; Aymamí, J.; Rodger, A.; Hannon, M.J.; Coll, M. “Molecular recognition of a three-way DNA junction by a metallo-supramolecular helicate”, Angewandte Chemie 2006, 45,1227−1231 (4) Dafforn, T. R.; Rodger, A. COSB 2004, 14, 541; Marrington, R.; Dafforn, T.R.; Halsall, D.J.; Hicks, M.; Rodger, A. “Validation of new microvolume Couette flow linear dichroism cells” Analyst, 2005, 130, 1608−1616


Project title: Peptidoglycan structure and reactions __________________________________________


Department: Chemistry _____________________________ Building, Room: B607 ____________________

E-mail address: [email protected]_______________ Phone number: 07876218199/48199/74696/23234


support to the supervisor and also agrees to meet the student briefly at least once a week): Name: __________________________________________

Department: _____________________________________ Building, Room: _________________________


Project outline:

A. Background to project

With the increasing advent of resistance to available drugs (including vancomycin, the antibiotic of ‘last resort’) we need to find antibiotics which circumvent the resistance mechanisms developed by bacteria. Hospital-acquired infections by multiply resistant organisms apparently cost the NHS >£1bn affecting more than 100,000 patients and causing over 5,000 deaths pa (NAO) in the UK alone. This project will focus on the structure and interaction with other molecules of the peptidoglycan layer which is embedded in the envelope of bacterial cells. Peptidoglycan forms a single, huge macromolecule with the size and shape of the cell. It is essential to bacteria and hence a potential targets for drug design, however, we know comparatively little about it structures as there is no ideal technique for studying its structures and dynamic rearrangements in solution.1,2


Peptidoglycan is a sugar/amino acid polymer that forms a mesh-like layer outside the plasma membrane of bacterial cells. Its biosynthesis is the site of action for several antibiotics, including the penicillin and cephalosporin β-lactams and the vancomycin group of glycopeptides. An alternative modus operandi is to target the peptidoglycan once formed, as the enzyme lysozyme does. Using ultra violet flow linear dichroism we have shown that the peptidoglycan can be flow oriented and that we can probe its degradation by lysozyme (Figure 1). In this project we will build on that preliminary discovery to understand more about the peptidoglycan layer. The aim is to probe independently the sugar and the amino acids. We will begin by collecting linear dichroism (LD), circular dichroism (CD) and absorbance data on the peptidoglycan itself. Then we will use this as a baseline to determine whether peptidoglycan binding ligands (e.g. methyl violet) bind in a geometrically organised manner. We will also probe the effect of lysozyme on peptidoglycans with and without ligands to determine how its lytic activity is affected by ligands.








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The project will then proceed in one of two directions. In the first we will look to developing new methods for studying peptidoglycan systems. We will choose a limited number of molecular systems from the first part of the project and determine what data can be obtained from infrared, oriented infra red, fluorescence detected linear dichroism and Raman methodologies.In an ideal world we may be able to examine single bond signals to enable the binding site (sugar or specific peptide) for a putative or actual drug to be determined, thus enabling predictions of potential resistance mechanisms and strategies for co-administering drugs to avoid resistance. The alternative path will be to investigate the mechanism of lysozyme activity by studying its modes of action on different timescales and concentration regimes.


The project will require laboratory sample handling of peptidoglycans, proteins and small molecules that bind to the peptidoglycan layer. It will involve the use of a range of spectroscopic and related techniques including LD, CD, DLS, , infra red absorbance, Raman,UV-visible absorbance and maybe fluorescence if we decide to include small molecule or protein fluorophores. Analysis of spectroscopic data to give steady state structures and kinetic parameters will form part of the project. The project will take place in collaboration with David Roper in Biological Sciences and Waldemar Vollmer from Newcastle.


In the first instance this project will cost ~£150. This project may involve visiting Newcastle to isolate more of the peptidoglycan if we use our current supplies.

E. Outline of a literature review, including starting references The literature review has two parts, one based on the peptidoglycans and one on the techniques to be used. It is suggested more effort is put into the former as the latter is often best first learned by trying to use the equipment then subsequently reading the literature. These references indicate the beginning of a reference trail. Peptidoglycan literature (1) Vollmer, W. and Höltje, J-V, ‘The Architecture of the Murein (Peptidoglycan) in Gram-Negative Bacteria: Vertical Scaffold or Horizontal Layer(s)?’ J. Bacteriology, 2004, 186, 5978-5987. (2) Vollmer, W. ‘Structural variation in the glycan strands of bacterial peptidoglycan’ FEMS Microbiol. Rev. 2008, 32, 287-306 Technique literature (1) Dafforn, T. R.; Rodger, A. COSB 2004, 14, 541; (2) Rodger, A. “Linear dichroism techniques” in Modern Techniques for Circular Dichroism Spectroscopy, Wallace, B.A. and Janes, R. (eds), IOS Press, Amsterdam, 2009 (3) Rodger, A.; Nordén, B. “Circular dichroism and linear dichroism”; Oxford University Press, 1997, pp150 (4) Rodger, A.; Marrington, R.; Geeves, M.A.; Hicks, M.; de Alwis, L.; Halsall, D.J.; Dafforn, T.R. “Looking at long molecules in solution: what happens when they are subjected to Couette flow?” Physical Chemistry Chemical Physics, 2006, 8, 3161−3171 (5) Marrington, R.; Dafforn, T.R.; Halsall, D.J.; Hicks, M.; Rodger, A. “Validation of new microvolume Couette flow linear dichroism cells” Analyst, 2005, 130, 1608−1616 (6) Hicks, M.R.; Rodger, A.; Thomas, C.M.; Batt, S.M.; Dafforn, T.R. “Restriction enzyme kinetics monitored by UV linear dichroism” Biochemistry 2006, 45, 8912−8917

Figure 1. Peptidoglygan UVLD measured on Jasco J-810. Green (lower LD trace) after the addition of lysozyme.


Project title: T cell antigen receptor membrane peptide: structure, function and activity mechanism _______


Department: Chemistry ____________________________ Building, Room: B607 ____________________

E-mail address: [email protected] ______________ Phone number: 07876218199/48199/74696/23234





Project outline:

A. Background to project Membrane proteins are either peripherally associated with or embedded within the cell’s membrane bilayer. They play roles in cell adhesion, cell growth and transport and in disease (infection and cure). Despite their importance, we still have only a limited understanding of their structures, function, and interactions with other molecules. At Warwick we have been developing UV-visible linear dichroism (LD) in concert with other techniques such as circular dichroism (CD), dynamic light scattering (DLS), fluorescence and absorbance, to probe the structure and kinetics of insertion of peptides and proteins interacting with model membranes: liposomes.1-5 Membrane protein systems are extremely important but very difficult to characterize and our facilities are unique in the world for this purpose.

This project will focus on a membrane peptide nonapetide (denoted CP, GLRILLLKV) and some mutants. CP is derived from the T cell antigen receptor (TCR) and has been shown to inhibit IL-2 production in T-cells following antigen recognition.6 In this way it inhibits the immune response. When administered subcutaneously or intraperitoneally CP reduces the induction of T-cell mediated inflammation in animal models with adjuvant induced arthritis. More recently it has been shown that CP and some of its conjugates are comparable in effect to cyclosporine, a clinical immunosuppressant, in reducing the acute phase of inflammation in the adjuvant induced arthritis models. CP has also been shown to be effective in humans and rodents as a treatment for psoriasis and contact dermatitis.7 Its administration via injection of transduced dendridic cells has been shown to induce antigen-specific immunosuppression in vivo.8


There is evidence to suggest that CP’s primary mode of action is via electrostatically disrupting the cell membrane. However, little is known about how it interacts with any of the molecules that will determine its mode of action. The aim of this project is to investigate the structure and intermolecular interactions of ** Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. However, all marks must be submitted at the latest by Sep 21 2009, 12 noon.







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CP as a function of environment. The ultimate goal is to improve its efficacy. Its structure in aqueous buffer, a range of simple organic/aqueous mixtures and detergents will be studied prior to investigating its behaviour when mixed with lipids. Most of the work will focus on model bilayer membrane environments (liposomes) where the nature of the surface and membrane flexibility will be varied by changing the lipid composition of the membrane. Circular dichroism (CD) will be used to probe the secondary structure of the peptide. There is evidence of random coil, α-helical, and β-structures in different environments. Linear dichroism (LD) is uniquely able to determine whether the peptide actually inserts into the membrane or merely interacts with the surface. The difference between activity and inactivity is often the timescale over which an interactions takes place—too fact may indiscriminately destroy cells and cause problems, too slow and no effect is noticed. Kinetic studies of the binding and putative insertion process will be studied by combining CD and LD to get structure and insertion data simultaneously.3

The two polar amino acids (near opposite ends of the peptide) of CP are known to be critical for receptor assembly. Mutations with different arrangements of these amino acids and substituting the shorter non polar alanine for non polar leucines will also be considered, if time allows, to determine what effect the different residues have.


The project will require laboratory sample handling of peptides and lipids, preparation of liposomes, use of a range of spectroscopic and related techniques including LD, CD, DLS, absorbance and maybe fluorescence if we decide to mutate in a tryptophan. Analysis of spectroscopic data to give steady state structures and kinetic parameters will form part of the project. It may also be possible to get structures of intermediates following the approach taken in reference 5. The project will take place in collaboration with Janice Aldrich-Wright, University of Western Sydney and colleagues at Westmead Hospital, Sydney.


The peptides and lipids will all be bought for the project which has the advantage of ensuring the project will achieve results and the disadvantage of being expensive. Initial expenditure will be of the order of £300. If the project proceeds particularly well and a number of mutants are required, a special case will be made for more resources.

E. Outline of a literature review, including starting references The literature review has two parts, one based on the techniques to be used and one on the peptide and its role in biological systems. It is suggested more effort is put into the latter as the former is often best first learned by trying to use the equipment then subsequently reading the literature. These reference indicate the beginning of a reference trail. Technique literature (1) Dafforn, T. R.; Rodger, A. COSB 2004, 14, 541; (2) Rodger, A.; Rajendra, J.; Marrington, R.; Mortimer, R.; Andrews, T.; Hirst, J. B.; Gilbert, A. T. B.; Halsall, D.; Dafforn, T.; Ardhammar, M.; Nordén, B.; Woolhead, C. A.; Robinson, C.; Pinheiro, T.; J., K.; Seymour, M.; Perez, N.; Hannon, M. J. In Biophysical Chemistry: Membranes and Proteins, ; Templer, R. H., Leatherbarrow, R., Eds.; The Royal Society of Chemistry: Cambridge, 2002, 3; (3) Hicks, M.R.; Damianoglou, A.; Rodger, A.; Dafforn, T.R.; “Folding and membrane insertion of the pore-forming peptide gramicidin occurs as a concerted process“ Journal of Molecular Biology, 2008, 383, 358-366 (4) Rajendra, J.; Damianoglou, A.; Hicks, M.; Booth, P.; Rodger, P. M.; Rodger, A. Chem. Phys. 2006, 326, 210; (5) Ennaceur, S.M.; Hicks, M.R.; Pridmore, C.J.; Dafforn, T.R.; Rodger, A.; Sanderson, J.M. “Peptide Adsorption to Lipid Bilayers: Slow Processes Revealed by Linear Dichroism Spectroscopy” Biophysical Journal, 2009, 96, Peptide literature examples (6) Manolios, N.; Collier, S., Taylor, J.; Pollard, J.; Harrison, L.; and Bender, V. Nature Med. 1997, 3, 84–88. (7) Gollner, G.P.; Muller, G.; Alt, R.; Knop, J.; and Enk, A.H. Gene Therapy 2000, 7, 1000-1004 (8) Mahnke, K.; Qian, T; Y.; Knop, J.; and Enk, A.H. Nature Biotech. 2003, 21, 903-908

H+A-

HA

HA H+ + A-

Confocal lens

Lipid bilayer

UME

H2O O2 A-

A-


Project title: Quantitative visualization of passive permeation across model cell membranes

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Pat Unwin _____________________________

Department: Chemistry ___________________________ Building, Room: Chemistry C526___________

E-mail address: [email protected] _____________ Phone number: 23264 ___________________





Project outline Background/rationale Cell membranes act as a semipermeable barrier to control the contents of a cell with respect to the surrounding environment and maintain homeostasis.1 Both passive (concentration driven) and active transport are important for the regulation of a cell. Among those molecules transported by passive permeation, (neutral) weak acids and bases are a significant class: there are a number whose pharmacological action depends on reaching specific intracellular sites of action; while others may exert detrimental effects on cellular processes. The rate of transport of such molecules is generally considered to be controlled by membrane solubility,2 but our recent work casts significant doubt on this hypothesis.3 This project will develop our recent work and provide much needed further examples to elucidate the factors that control membrane permeation. Programme

To study passive permeation requires that the permeant is delivered to the membrane in a well-defined and quantifiable way. This has not been accomplished in conventional techniques, and so we have developed a method based on ultramicrodes (UMEs) and laser scanning confocal microscopy (LSCM). By positioning an UME close to a membrane surface and using it to carry out the electrogeneration of protons (or hydroxide ions), one can impose a well defined local flux of the desired species. The current at the electrode can be tuned to precisely control the proton/hydroxide generation rate. In the case shown left, electrogenerated protons combine with the conjugate anion in solution, making a weak acid which may be transported across the membrane. By mapping the pH in the region of the electrode and membrane (on both sides) one can readily deduce the pathways of the protons. pH mapping on a




(8 weeks) x Biophysical chemistry x Slot 2 (18/05 to 10/07/08) Submission of thesis by: 8.59 am 13/07/08; viva to be scheduled in agreement with supervisor

Mathematics/computing x Slot 3 (13/07 to 04/09/08) Submission of paper by: 8.59 am 7/09/08 Talk: 3/10/08, MOAC Seminar Room

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rapid timescale will be achieved using high speed LSCM, with pH sensitive fluorophores in solution and located within the membrane. These studies will examine a range of planar lipid bilayer membranes (formed either in a Teflon sheet3 or at the end of a glass capillary4) and investigate how bilayer composition (e.g. the effect of cholesterol, lipid mixture) influences transport rates. A wide range of weak acids can be investigated, including those of pharmacological significance and others which will constitute a family of molecules to enable us to build a comprehensive understanding of structure-activity relationships. The molecules to be studied will be decided in consultation with the student. To obtain quantitative information required modelling of the pH maps produced and we have codes available for use by those who are interested in this aspect. The project is potentially vast in scope and this mini-project will naturally look at just one or two molecules, while providing a “taster” of a very powerful technique that we believe will also be applicable to active transport processes and living cells. The project addresses the core aim of MOAC, i.e. understanding the structure and function of biomolecular assemblies (in this case lipid bilayers) and has at its heart the development/application of new instrumentation and techniques. There would be the option for a PhD project to develop along these lines (see next section). Related Staff and Miniprojects This miniproject is closely related to the Maths/computing project “Passive transport of weak acids across lipid bilayers: insights from molecular dynamics” supervised by Prof. Mark Rodger and we expect that both aspects could develop into a PhD project under the joint supervision of Profs. Rodger (Chemistry) and Allen (Physics), on computational aspects, and Unwin on experimental aspects. We believe the combination of experimental methods and simulation on the same well-designed systems will provide key new insights into the nature of membrane transport. Skills to be learnt The project will provide experience in micro-nanoscale quantitative methods, notably ultramicroelectrodes, scanning electrochemical microscopy and laser scanning confocal microscopy. Additionally, there is the possibility of extensive data analysis with finite element modelling packages (although this in not the main aim of the project). The project will make use of facilities in the Warwick Electrochemistry and Interfaces Group (www.warwick.ac.uk/electrochemistry) and there will be the opportunity to interact with PhD students undertaking a wide range of projects in interfacial science. Timescale and resources Because we are developing a new confocal microscopy suite in Chemistry which will ready at the end of April 2009, this project needs to run in either slot 2 (preferably) or slot 3. The project will require resources of ca. £200 to cover lipids, fluorophores, metals for electrodes etc. References 1. R. B. Gennis, Biomembranes, Springer, New York, 1989. 2. See for example: Q. Al-Awqati, One hundred years of membrane permeability: Does Overton still rule? Nat. Cell Biol. 1 (1999) E201–E202; A. Walter and J. Gutknecht, Monocarboxylic acid permeation through lipid bilayer membranes. J. Membr. Biol. 77 (1984) 255–264. 3. J. M. A. Grime, M. A. Edwards, N. C. Rudd and P. R. Unwin, Quantitative visualization of passive transport across bilayer lipid membranes Proc. Natl. Acad. Sci. USA, 105 (2008) 14277–14282. 4. B. Zhang, J. Galusha, P. G. Shiozawa, G. Wang, A. J. Bergren, R. M. Jones, R. J. White, E. N. Ervin, C. C. Cauley, and H. S. White, A Bench-Top Method of Fabricating Glass-Sealed Nanodisk Electrodes, Glass Nanopore Electrodes, and Glass Nanopore Membranes of Controlled Size, Anal. Chem., 79 (2007) 4778-4787. Futher references available on request.


Project title: Development of electrochemical methods to facilitate real-time amperometric detection of catecholamine neurotransmitters in hippocampal brain slices

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Pat Unwin and Bruno Frenguelli____________

Departments: Chemistry and Biological Sciences _______ Building, Room: Chemistry C526 (PRU) _____

E-mail address: [email protected] _____________ Phone number: 23264 (PRU)______________





Background and aims of the project The mammalian hippocampus is the seat of several forms of learning and memory, in particular the memory of events (episodic memory) and places and environments (spatial memory). The forgetfulness of Alzheimer's Disease is reflected in the damage that such patients suffer to the hippocampus and associated structures. The cellular and molecular processes involved in these forms of memory can be studied in whole animal models and in isolated hippocampal tissue. One of us (BF) has long experience in working with hippocampal "slices" - thin (400 µm) cross sections of the rodent hippocampus (Fig 1) - that can be maintained in vitro for several hours and from which real-time electrophysiological, optical and amperometric recordings can be made. Previously BF, in collaboration with Prof Nick Dale, has used enzyme-based biosensors for ATP, adenosine and glutamate in models of seizure activity and cerebral ischemia, insults to which the hippocampus is especially vulnerable. However, in the context of learning and memory, and the cellular model for this - synaptic plasticity - it is clear that the catecholamines noradrenaline (NA) and dopamine (DA) play a major regulatory role. NA and DA are believed to convert transient synaptic signals into persistent, protein synthesis-dependent memories. Thus, their release, inferred indirectly via pharmacological antagonism, gates the transition from short-term to long-term memory, but due to the fleeting nature of their presence in the synapse, their release has yet to be detected directly.




(8 weeks) x Biophysical chemistry x Slot 2 (18/05 to 10/07/08) Submission of thesis by: 8.59 am 13/07/08; viva to be scheduled in agreement with supervisor


Fig 1. Dopaminergic and noradrenergic projections to the hippocampus. The hippocampus is composed of areas CA1, CA3 and DG (dentate gyrus).

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We intend to develop state-of-the-art amperometric techniques which will enable us to measure directly the release of NA and DA during electrical stimulation of synapses. We will use knowledge, of the type displayed in Fig 1, to position electrodes in discrete locations where the largest DA or NA signals might be expected. In the longer term this would provide a means of equating the kinetics of DA/NA release with the persistence of electrophysiological changes at the synapse. Programme While there has been good progress in the use of carbon fibre electrodes for amperometric detection of catecholamines in brain slices1 and at the level of single neurons,2 amperometry at this type of electrode exhibits large background currents so that significant correction procedures are necessary in the interpretation of data, which potentially restricts information content. At Warwick, we have been pioneering the use of new forms of carbon, such as single-walled carbon nanotubes (SWNTs)3,4 and conducting diamond5 as electrode materials with greatly improved properties. In the present project we will assess carbon nanotube network electrodes for the detection of DA and NA, as these electrodes appear to be particularly sensitive for amperometric detection. We have shown, for example, that SWNT electrodes offer unprecedented sensitivity for trace level detection with cyclic voltammetry3 and that they are scalable to microelectrode formats.4 A key aspect of this project will involve the optimisation of this type of electrode to allow deployment in scanning electrochemical microscopy (SECM).6 The use of SWNT microelectrode probes in SECM will allow us to make spatially-resolved measurements on a rapid timescale. This will require identifying limits of detection in complex media, and the ability to identify specific chemical components. Final investigations will assess the use of the electrodes in measurements of DA and NA at discrete locations, as described above. Modelling of the electrode responses is a further option, which will draw on our expertise in mass transport modelling/interfacial kinetics.7 Location of the project and skills development The project will be based in Chemistry and will involve electrochemistry, microfabrication, and electrode characterization. In Chemistry, the project will be supervised by Pat Unwin and there will also be strong collaboration with Julie Macpherson. Measurements on brain slices will be in Biological Sciences, under the supervision of Bruno Frenguelli. Both research groups are exceptionally well resourced and have state of the art facilities. There will be the opportunity to interact with, and learn from, PhD students undertaking a wide range of projects in interfacial science (e.g. www.warwick.ac.uk/electrochemistry). The project is potentially vast in scope and this mini-project will provide an introduction to potentially powerful techniques that could have wide application in the life sciences in the longer term. Timescale and resources This project needs to run in either slot 2 or slot 3. The project will require resources of ca. £200 to cover part of the consumables costs. Other costs will be provided by the research groups involved. References 1. B. J. Venton and R. M. Wightman, Anal. Chem., 2003, 414, 414A. 414 (2003) 414A. 2. See for example: S. E. Hochstetler, M. Puopolo, S. Gustincich, E. Raviola, and R. M. Wightman, Anal. Chem., 2000, 72, 489-496 3. P. Bertoncello, J. P. Edgeworth, J. V. Macpherson and P. R. Unwin, J. Am. Chem. Soc. 2007, 129, 10982–10983. 4. I. Dumitrescu, P. R. Unwin, N. R. Wilson and J. V. Macpherson, Anal. Chem., 2008, 80, 3598–3605. 5. L. Hutton, M. E. Newton, P. R. Unwin and J. V. Macpherson, Anal. Chem., 2009, 81, 1023–1032. 6. See for example: S. Amemiya, A. J. Bard, F-R. F. Fan, M. V. Mirkin and P. R. Unwin, Annu. Rev. Anal. Chem., 2008, 1, 95–131. 7 See for example: M. Gonsalves, A. L. Barker, J. V. Macpherson, P. R. Unwin, D. O'Hare and C. P. Winlove, Biophys. J., 2000, 78, 1578-1588.


Project title: Structural Analysis of the Twin Arginine Translocase Pathway Protein TatA using Synthetic Peptides

Supervisors (the person who will be doing the day to day supervision of the mini-project):

1. Name: Ann Dixon_______________________________

Department: Chemistry_______________________ Building, Room: Physical Sciences, C503 ____

E-mail address: [email protected] _______ Phone number: 50037 ___________________

2. Name: Colin Robinson___________________________

Department: Biological Sciences _______________ Building, Room: Biological Sciences ________

E-mail address: [email protected]_ Phone number: 23557 ___________________

Background. The Twin-arginine translocase (Tat) pathway is involved in the export of folded proteins from thecytoplasm to the periplasm [1]. In Escherichia coli three membrane proteins essential for the Tat pathway havebeen identified; TatA, TatB and TatC. TatB and TatC form a complex with small amounts of TatA, however themajority of TatA forms separate, homo-oligomeric complexes varying in size from less than 100 kDa to over 500kDa [2, 3]. It has been proposed that these TatA complexes form the pore through which proteins are translocated.TatA itself is a relatively small protein, 9.6 kDa, and is predicted to have an N-terminal α-helical transmembranedomain, followed by an amphipathic α-helical domain, and a non-essential largely unstructured C-terminus (Figure1) [4]. It is highly unusual for a single span membrane protein to form such a wide variety of different sizedcomplexes, and the exact mechanism by which TatA complexes assemble is still unknown. Transmembranedomains have been shown to be involved in the formation of protein complexes [5] and previous studies suggestthat the TatA transmembrane domain may be involved in complex formation [6, 7].

Figure 1. Predicted secondary structure of TatA. N-terminal transmembrane (TM) domain, followed by an α-helical amphipathichelix (APH) and a largely unstructured C-terminal

The importance of the amphipathic helix has also yet to be determined. It is known to be important for translocation[4, 6] and it has been suggested that it inserts into the membrane as part of the translocation process [8]. Thispossible insertion could lead to interactions between the transmembrane domain and amphipathic helix in themembrane.





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Aims of project. This project involves the analysis of synthetic peptides corresponding to the TatA transmembranedomain and the amphipathic helix. Synthetic peptides have been widely used to characterise individual proteindomains and can provide information which is difficult to obtain from the full-length protein. We aim to use a varietyof biophysical techniques to fully characterise the TatA transmembrane domain and amphipathic helix peptides indetergent micelles (commonly used to solubilise membrane proteins) and liposomes (which represent moreaccurately natural membrane environments).

Previous work has shown that the transmembrane domain peptide is able to insert into lipid bilayers and can formSDS-stable dimers, however it’s interaction with the amphipathic helix has yet to be analysed. The secondarystructure of the amphipathic helix peptide has been shown to vary depending on detergent concentration and thelipid composition of liposomes.

Programme of work. Peptides corresponding to the transmembrane domain and amphipathic helix of TatA will bepurified using reversed-phase HPLC, and their purity will be confirmed using mass spectrometry. The secondarystructure and thermal stability in a range of detergent micelle concentrations, and liposome compositions will bemeasured using circular dichroism. Insertion of the peptides into lipid membranes will be analysed by lineardichroism. Homo- and hetero- interactions between the two peptides will be probed using SDS-PAGE and chemicalcross-linking in detergent micelles. Time permitting the ability of the peptides to form homo- and hetero-oligomerswill also be studied using analytical ultracentrifugation.

Resources required.£200 for detergents, lipids and HPLC solvents.

Outline of a literature review. Structure and function of Tat proteins Role of TatA in translocation State of knowledge on structure of TatA Use of circular dichroism, linear dichroism and analyical ultracentrifugation for studying membrane proteins

Starting references.1. Robinson, C. and A. Bolhuis, Protein targeting by the twin-arginine translocation pathway. Nat Rev Mol Cell Biol, 2001.

2(5): p. 350-6.2. Gohlke, U., et al., The TatA component of the twin-arginine protein transport system forms channel complexes of

variable diameter. Proc Natl Acad Sci U S A, 2005. 102(30): p. 10482-6.3. Oates, J., et al., The Escherichia coli twin-arginine translocation apparatus incorporates a distinct form of TatABC

complex, spectrum of modular TatA complexes and minor TatAB complex. J Mol Biol, 2005. 346(1): p. 295-305.4. Lee, P.A., et al., Truncation analysis of TatA and TatB defines the minimal functional units required for protein

translocation. J Bacteriol, 2002. 184(21): p. 5871-9.5. Lemmon, M., et al., Glycophorin A dimerization is driven by specific interactions between transmembrane alpha-

helices. J. Biol. Chem., 1992. 267(11): p. 7683-7689.6. Greene, N.P., et al., Cysteine Scanning Mutagenesis and Disulfide Mapping Studies of the TatA Component of the

Bacterial Twin Arginine Translocase. J. Biol. Chem., 2007. 282(33): p. 23937-23945.7. Porcelli, I., et al., Characterization and membrane assembly of the TatA component of the Escherichia coli twin-

arginine protein transport system. Biochemistry, 2002. 41(46): p. 13690-7.8. Gouffi, K., et al., Dual topology of the Escherichia coli TatA protein. J Biol Chem, 2004. 279(12): p. 11608-15.


Project title: Monitoring Membrane Protein Interactions using Fluorescence Spectroscopy


Name: Ann Dixon_________________________________

Department: Chemistry_____________________________ Building, Room: Physical Sciences, C503 ____


Supervisor’s advisor:

Name: Alison Rodger

Department: Chemistry ____________________________ Building, Room: Physical Sciences, B607 ____

E-mail address: [email protected] _____________ Phone number: 23234___________________

Background. Structural, biophysical and biochemical studies have revealed the importance of membrane proteinsin orchestrating a vast array of biological processes. These proteins are found embedded in membranes that bothsurround and permeate cells, where they perform functions crucial to the survival of individual cells (e.g. import/export of nutrients and waste, energy production, etc.) and to the myriad of systems present in multicellularorganisms (e.g. hormone signalling, immune response, etc). To date, membrane proteins have been foundprimarily in two structural types where the membrane spanning region, better known as transmembrane domain(TM), is composed of either (a) single/multiple α-helices or (b) multiple beta sheets that form a barrel-like structure(Figure 1).

(a) (b)Figure 1. Representative structures of a) α-helical and b) β-barrel membrane proteins. Yellow band represents the membranebilayer.

The lateral association of multiple membrane spanning -helices in the membrane bilayer is believed to play a keyrole in the structure and function of helical membrane proteins. One such case is the major histocompatibilitycomplex (MHC) class II-associated invariant chain (Ii). Ii is a component of the mammalian immune system andresponsible for the correct assembly of MHC Class II (MHC) proteins that activate T-cells and hence elicit animmune response to infection. Shortly after biosynthesis, Ii self-associates to form a homotrimer and then bindsthree hetero-dimers of MHC to form a nine chain complex in the endoplasmic reticulum (ER) (Figure 2). It is onlywhen part of this complex that MHC can be released from the ER to take part in the antigen presentation pathway.

It had been proposed that the -helical TM domain plays a key role in the trimerization of Ii, providing the first stepin this antigen presentation pathway. Sequence analysis and mutagenesis studies on Ii have revealed conserved









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polar residues (Glu47, Thr50) within the TM domain that are vital for maintaining function of the full length protein,possibly through the formation of interhelical hydrogen bonds. The effect of mutating these polar residues on theoligomeric state of the Ii TM domain (and ultimately the structure of Ii) is unknown and will be the focus of thisproject. Building upon work all ready performed for the wild type Ii TM domain, the student will use fluorescenceresonance energy transfer (FRET) analyses to study the effect of mutations G47A and T50A on self-association ofthe TM domain.

Figure 2. Proposed structure of Ii (blue) complex with MHC (grey). Helical transmembrane domains are depicted as longcylinders.

FRET is commonly used for studying protein-protein interactions, but there has been relatively little application of itto the study of membrane proteins and in particular the interactions of TM domains. The technique relies on thedistance-dependent transfer of energy between a donor fluorophore (e.g fluorescein) and an acceptor fluorophore(e.g rhodamine) which is observed as a reduction in the fluorescence emission of the donor and an increase in theemission of the acceptor. Both of these fluorophores can be conjugated to synthetic peptides using standardmethods. In addition to FRET, the student will use HPLC to purify flourophore-labelled synthetic peptides, chemicalcross-linking in conjunction with SDS-PAGE to assess oligomeric state, mass spectrometry to assess purity ofpeptides and circular dichroism to assessing secondary structure. All necessary materials and equipment areavailable within the Department of Chemistry.

Programme of work. Fluorophore-tagged peptides corresponding to the mutated transmembrane domain of Ii willbe purified using reversed-phase HPLC, and their purity will be confirmed using mass spectrometry. The secondarystructure in a range of detergent micelle concentrations will be measured using circular dichroism. Self-associationof the mutant Ii peptides will be probed using SDS-PAGE and chemical cross-linking in detergent micelles, followedby FRET measurements of donor and acceptor-labeled peptides at a variety of detergent concentrations.

Resources Required.£200 for HPLC solvents, detergent stocks and consumables for SDS-PAGE.

Outline of literature review.

Structure and function of -helical membrane proteins Role of sequence motifs in stabilizing transmembrane domains State of knowledge on structure and function of MHC Class II-associated invariant chain Use of fluorescence techniques for studying protein-protein interaction in TMDs

Starting references.

1. Dixon, A. M.; Stanley, B. J.; Matthews, E. E.; Dawson, J. P.; Engelman, D. M., Invariant chain transmembranedomain trimerization: A step in MHC class II assembly. Biochemistry 2006, 45, (16), pp. 5228-5234.

2. Johnson, A. E., Fluorescence approaches for determining protein conformations, interactions and mechanismsat membranes. Traffic 2005, 6, (12), pp. 1078-92.

3. Fisher, L. E.; Engelman, D. M.; Sturgis, J. N., Detergents modulate dimerization but not helicity, of theglycophorin A transmembrane domain. Journal of Molecular Biology 1999, 293, (3), pp. 639-651.

4. MacKenzie, K. R.; Fleming, K. G., Association energetics of membrane spanning alpha-helices. Curr Opin StructBiol 2008, 18, (4), pp. 412-419.

5. You, M.; Li, E.; Wimley, W. C.; Hristova, K., Forster resonance energy transfer in liposomes: measurements oftransmembrane helix dimerization in the native bilayer environment. Anal. Biochem. 2005, 340, (1), 154-164.


Project title: Hormone binding analysis of a putative ABA receptor protein

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Richard Napier (with Andrew Marsh in Chemistry [email protected], ext 24565 and Martin Sergeant in

Warwick HRI)

Department: Warwick HRI ______________________ Building, Room: Wellesbourne PP 1.29

E-mail address: [email protected] _________ Phone number: ext 75094

Project outline: Background: The hormone abscisic acid (ABA) regulates both germination and water balance in plants and the identification of the receptor for ABA is a prime target for developmental biologists. In 2006 two receptors for ABA were published (Razem et al, Nature 2006, 439:290; Shen et al, Nature 2006, 443:823) with a further one in 2007 (Liu et al, Science 2007, 315:1712). All three publications identified different proteins. All three have since been withdrawn or seriously doubted. The Warwick labs of Marsh and Napier developed Magic Tag® technology in order to identify novel binding partners (Dilly et al, Chemical Communications 2007, 27:2808). Also the basis of a successful spin-out company, the technique was combined with protein library display on viral particles. Using ABA as an immobilized ligand we selected putative ABA-binding proteins from the library, one in particular appeared especially attractive on the basis of bioinformatics work. Several hits came through secondary screening and two have been cloned into bacterial expression vectors and shown to over-produce soluble protein (M Sergeant, A Thompson unpublished). The Project will use these cell lines to express the proteins from the viral screens. The proteins will be purified using FPLC by means of an epitope tag inserted during cloning. Using negative and positive controls from the lab, you will use different activity assays to test whether or not the proteins bind free ABA. This will be the next test for the hypothesis that our screen identified a putative, novel ABA receptor. One set of assays will be radioligand binding assays, by precipitation and/or by equilibrium dialysis. The other will be to use either surface plasmon resonance (Biacore SPR) to test for binding to a well characterized biotinylated ABA conjugate, or isothermal calorimetry (ITC) (Risk et al. Nature 456, 11 Dec, E5) to test for binding to free ABA in solution. The method chosen will depend on the quantity of protein purified. All the binding data will be compared to the kinetics of the positive control, a cell line expressing a monoclonal antibody to ABA that we have characterized extensively. Skills to be learnt: protein expression, protein purification, small molecule binding assays, binding kinetics, affinity measurement, thermodynamics Costs: No more than £450, to include reagents, column use, Biacore chip







Mathematics/computing possibly Slot 3 (13/07 to 04/09/08)

Submission of paper by: 8.59 am 7/09/08 Talk: 3/10/08, MOAC Seminar Room

B3 and PS10

*

*

*

*

T r i t icum ae s t i v um(I / I I) ------MG--C---DDKCG---CAVP--CPGGTGCRCT 22

P ice ag l a uca ------MSGTC-TRGPACG---CGET--CACVD-CKCG 25Pseudo t sugamenz i e s i ------MASTC-TRGAECG---CGET--CACVD-CKCG 25La r i xkaempefe r i ------MASTC-TRGPECG---CGET--CACVD-CKCG 25P i n u st aeda ------MSS---TRSPQCG---CGET--CACAD-CKCG 23Chamaecypa r i sob t u s a ------MEGTCGCVATHCG---CLANGNCTCST-CRCA 28Cryptomer i aj apon ica ------MEGSCACVTSSCG---CLTNGNCTCDT-CKCA 28

* ** * * *:*

MOAC mini-project proposal Submit up to TWO pages including this header page to http://www2.warwick.ac.uk/fac/sci/moac/intranet/miniproj/mp_proposal_submission_form_2008_9 by 9 February 2009. Any queries contact Mónica Lucena [email protected]. Project title: Investigating the structural basis of metal binding and dynamics in metallothionein clusters – Part 2

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Dr Oksana Leszczyszyn

Department: Chemistry ____________________________ Building, Room: Chemistry, A112



support to the supervisor and also agrees to meet the student briefly at least once a week): Name: Dr Claudia Blindauer

Department: Chemistry ____________________________ Building, Room: Chemistry, C510


Project outline: Background and Motivation. Metallothioneins (MTs) are small, cysteine-rich metalloproteins. These diverse proteins bind large amounts of both essential and non-essential metal ions such as Zn, Cu, Cd, Hg and As, in specialised metal-thiolate structures known as clusters. The most well-characterised MT domains are α and β clusters, which bind four and three metals respectively. The Blindauer group has recently elucidated the solution structure of a new cluster expressed in a MT from common wheat (see figure). This binuclear cluster will be the focus of this study.

Bioinformatic analysis has identified over 50 homologous binuclear domains in a diverse array of plant species. These homologues can be mapped through time from the relatively young seeding plants (first identified in the fossil record ca. 400 million years ago) to older evolutionary branches such as mosses and liverworts. In the majority of these plant species (i.e. monocots and dicots) the positions of the key cysteine metal binding residues are highly conserved. However, in tree species significant variations in both the number and position of cysteines have been observed. Given that the number and arrangement of cysteines in MT clusters has been shown to have important implications for structure, metal binding stoichiometry and dynamics, it is hoped that this

work would shed light on this phenomenon and begin to answer the question of what biochemical advantages or

differences such structural adaptations would confer. Aim. This is the second part of two 8-week projects that fit into a wider research proposal which aims to investigate the effect that variations in cysteine number and position have on the stoichiometry, structure and metal binding ** Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. However, all marks must be submitted at the latest by Sep 21 2009, 12 noon.







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dynamics of MT clusters. The objectives of this part of the study focus on structural studies of four binuclear clusters from different plant species: (i) Triticum aestivum, (ii) Picea glauca, (iii) Pinus taeda and (iv) Chamaecyparis obtusa.

Programme of work. Structural studies require a multidisciplinary approach to solve challenging and interesting bioinorganic problems. Initially, the researcher will undertake ESI-MS to characterise the metal binding stoichiometry and selectivity of the four proteins described above. Following this, multidimensional proton NMR will be utilized to characterise the protein folds of the four domains. It is hoped that the protein samples for this will have been prepared in Part 1 of this study, but a small amount of protein purification may be required.

Skills to be learned. It is hoped that during this project you will develop skills in acquiring mass and NMR spectra of biological samples. Furthermore, you will be trained to interpret protein mass and NMR spectra. Resources required. A small budget will be required for consumables for NMR spectroscopy. This is estimated at approximately £100-£150. Recommended reading. O.I. Leszczyszyn, R. Schmid, C.A. Blindauer, 2007, Proteins, 68(4): 922-935 O.I. Leszczyszyn, C.D. Evans, S.E. Keiper, C.A. Blindauer, 2007, Inorganica Chimica Acta, 360(1):3-13 M.J. Cismowski, S.S. Narula, I.M. Armitage, Journal of Biological Chemistry, 1991, 266(36):24390-24397 E. Freisinger, 2008, Dalton Transactions, 47: 6663-6675 M.L. Chernaik, P.C. Huang, 1991, PNAS, 88(8):3024-3028


Project title: Structure/function studies on ALDC, part 1; Chemistry

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Professor Martin Wills

Department: Chemistry ___________________ Building, Room: Chemistry C block C504 ________

E-mail address: [email protected] _______________ Phone number: 024 7652 3260

Supervisor’s advisor (for non-permanent members of staff or those on probation: academic who agrees to provide supervision support to the supervisor and also agrees to meet the student briefly at least once a week): n/a.

Name:__________________________________________ Department: _____________________________________ Building, Room: ________________________ E-mail address: __________________________________ Phone number: ________________________ Project outline: A Background: This mini-project proposal is the first of three directed towards the study of the mechanism of action of the enzyme acetolactate decarboxylase (ALDC). The objective of this mini-project will be to prepare two substrate and transition state analogues for co-crystallisation with the isolated enzyme. The second mini-project will be the X-ray crystal structure analysis of ALDC bound to the analogues (Professor V. Fulop). The third mini-project will involve a molecular modelling study of the enzyme, (Professor P. M. Rodger).

Acetolactate decarboxylase (ALDC) catalyses the decarboxylation of (S)-α-acetolactate [(S)-2-hydroxy-2-methyl-3-oxobutanoate] 1 and (S)-α-acetohydroxybutyrate [(S)-2-ethyl-2-hydroxy-3-oxobutanoate] 2 (Scheme 1),1,2 the biosynthetic precursors of valine 3 and isoleucine, 4 (Scheme 1). The products of decarboxylation of 1 and 2 are (R)-acetoin 5 and (R)-3-hydroxypentan-2-one 6 respectively. The enzyme catalyses also the decarboxylation of the corresponding (R)-enantiomers. Decarboxylation of both enantiomers of α-acetolactate leads to a single (R)-enantiomer of 5, by the mechanism shown in Scheme 2, by first decarboxylating the (S)-enantiomer 1 with overall inversion of configuration at the α-centre and then by catalysing a tertiary ketol rearrangement of the (R)-enantiomer 7 with migration of the carboxylate group to give 8.3-4

B Programme The full length (260 amino acid residues) of ALDC from Bacillus brevis has been cloned, expressed and purified by Novozymes A/S (Denmark) to homogeneity and provided to us for crystallization trials.5 A major question concerns the location of the active site. It will be necessary to synthesise substrate or product analogues that can be co-crystallised for X-ray structural studies.

In this part of the three-part project, a series of synthetic inhibitor molecules shall be prepared for evaluation as active-site binders to the enzyme. Specific synthetic targets to be made in this project are shown in Figure 1. Analogues 9 should bind but cannot undergo decarboxylation. The use of product analogue diols of type 10 will also be explored. The nature of the binding shall provide information about the mechanism of the enzyme.





(8 weeks) X Biophysical chemistry (essentially Chemistry) Slot 2 (18/05 to 10/07/08)

Submission of thesis by: 8.59 am 13/07/08; viva to be scheduled in agreement with supervisor


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MeCO2

-O

R OHMe

R

OH1 R = Me2 R = Et

5 R = Me6 R = Et

ALDCR

CO2-

Me

NH2

3 R = Me4 R = Et

Scheme 1 - CO2

S

RO

OMe

CO2-

OMe HH+

OHMeOMe

CO2-

ADCScheme 2

78

RS

R1CO2H

OH

R2 OHR1

R2

OH

OH9 10

Figure 1: Potential analogues for co-crystallisation with ADC. (R1=Me or Et and R2=Me or Et)

C Skills In this section of this three part project: synthetic organic chemistry, manipulation of chemicals under inert conditions purification and characterisation methods including NMR, Mass spectrometry, gas chromatography and high performance LC (HPLC). A method has already been established for the small scale synthesis, in enantiomerically enriched form, of two derivatives of inhibitor 9. This method, which involves 2 steps from commercially available starting materials, shall be scaled up to provide sufficient quantities of required materials for co-crystallisation with the enzyme.

D. Consumables budget: £ 200 for reagents. E. References1). Dolin, M.I. and Gunsalus, I.C. (1951). J. Bact. 62, 199-214. 2). Løken, J.P. and Størmer, F.C. (1970). Eur. J. Biochem. 14, 133-137. 3). Crout, D.H.G. and Rathbone, D.L. (1988). J. Chem. Soc., Perkin Trans. 1, 98-99. 4). Crout, D.H.G., Rathbone, D.L. and Lee, E.R. (1990). J. Chem. Soc., Perkin Trans. 1, 1367-1369. 5) Najmudin, S., Andersen, J. T., Patkar, S. A., Borchert, T. V., Crout, D. H. G. and Fülöp, V. (2003). Acta. Cryst. D59, 1073-1075.


Project title: Discovery and Characterisation of Lignin-degrading Bacteria using a Fluorescence Assay

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Professor Tim Bugg (group member Mark Ahmad will provide laboratory assistance)

Department: Chemistry ____________________________ Building, Room: C513

E-mail address: [email protected] _____________ Phone number: ext. 73018

[part of collaboration with Prof. D. Pink & Dr. K. Burton, Warwick HRI, Dr. P. Norris & Prof. E. Wellington, Biological Sciences]

A. Background. Lignin is a structurally complex and heterogeneous aromatic polymer, it is one of the major components of plant biomass, comprising 10-30% by weight of cereals such as wheat straw. Breakdown of plant lignocellulose to glucose is a key step in second-generation biofuel production [1], but a current limitation in this process is the resistance of lignin to breakdown. TDHB’s research group has considerable experience in the enzymology of bacterial aromatic degradation [2], and we have recently developed a new fluorescence assay for lignin breakdown, by attachment of a fluorescent reporter group to milled wood lignin, which we have used to identify several strains of soil bacteria that are able to degrade lignin into low molecular weight fragments, using extracellular lignin peroxidase enzymes [3].


Project timing1


(8 weeks) X Biophysical chemistry X Slot 2 (18/05 to 10/07/08) Submission of thesis by: 8.59 am 13/07/08; viva to be scheduled in agreement with supervisor

Mathematics/computing X Slot 3 (13/07 to 04/09/08) Submission of paper by: 8.59 am 7/09/08 Talk: 3/10/08, MOAC Seminar Room

β-aryl ether phenylcoumarane

β-aryl ether (51%)

O RMeO O H OMe

O

O

OMe

OMe

O

O H OMe

OAr OMe

O H

OMeO H

R O

O

O HOMe

MeOOMeO

R OO HO

O H

OMe

OMe

H O

O HOMe

H O O

O H

C HO

MeO

O H

OMeO

O

OMeO H

O HOMe

O

H OOMe

C H2O H

OMe

H O

MeO C O2H

O H

H O2C

biphenyl

pinoresinol biaryl ether

phenylcoumarane (12%)

O HOMe

H O

biphenyl (11%)

Structures found in lignin:

diarylpropane (2%)

O H

O H

diaryl propane

OMe

pinoresinol (2%)

PS14

This mini-project will use this fluorescent assay to screen for further bacterial lignin degraders, and to characterize in more detail an existing set of bacterial degraders that we currently have. If a student wished to combine this mini-project with a 2nd mini-project in biological aspects of lignocellulose breakdown in Biological Sciences or Warwick HRI, that might be possible, subject to approval by MOAC, please contact Prof. Bugg to discuss further. B. Mini-project Programme. Milled wood lignin will be prepared using existing procedures from plant sources such as wheat straw, willow, and poplar. Samples of lignin will be labeled with fluorescent reporters such as fluorescein. The fluorescent lignin will be used in a time-based assay, using a micro-titre plate reader, to screen for new lignin degraders, using thermophilic bacteria from geothermal soils (from Dr. P. Norris, Biological Sciences) or strains of actinomycetes (from Prof. E. Wellington, Biological Sciences). The dependence of these strains on added hydrogen peroxide will be assessed using the assay, and the specificity of new degraders will be assessed, using lignins obtained from diverse plant sources. New degrading strains could then be tested in small-scale lignocellulose breakdown trial experiments. The fluorescent assay will also be used to characterize further a set of existing bacterial lignin degraders that we currently have. The dependence upon growth media, temperature, and pH will be studied, and whether the degrading enzymes are inducible by either lignin itself, or small molecule lignin fragments (available in group). C. Skills: The project will involve preparation & purification of fluorescent materials, microbiology, use of fluorescence spectroscopy, and enzyme kinetics. D. Consumables budget: £200 for fluorescence reagents. E. References [1] “Sustainable biofuels: prospects and challenges”, Report by the Royal Society, January 2008. [2] S. Mendel, A. Arndt, and T.D.H. Bugg, Biochemistry, 43, 13390-13396 (2004); J-J. Li, C. Li, C.A. Blindauer, and T.D.H. Bugg, Biochemistry, 45, 12461-12469 (2006); C. Li, J-J.Li, M.G. Montgomery, S.P. Wood, and T.D.H. Bugg, Biochemistry, 45, 12470-12479 (2006); M. Xin and T.D.H. Bugg, J. Am. Chem. Soc., 130, 10422 (2008). [3] R. Vicuna, Enz. Microb. Technol., 10, 646 (1988).

FITC-Lignin + cells: Fl change over 0-2 hr

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Figure 2. Assay of FITC-labelled lignin with bacterial cells, fluor change over 0-2 hr


Project title: Labelling of Bacterial Cells with Fluorescent Peptidoglycan Intermediates

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Professor Tim Bugg (group member Sandeep Sandhu will provide laboratory assistance)

Department: Chemistry ____________________________ Building, Room: C513

E-mail address: [email protected] _____________ Phone number: ext. 73018

[part of ongoing collaboration with Prof. C. Dowson & Dr. D. Roper, Biological Sciences]

A. Background. Bacterial cell wall peptidoglycan biosynthesis is the site of action of many clinically important antibiotics, including the beta-lactam family of penicillins, and the vancomycin group of glycopeptide antibiotics [1]. The biosynthetic pathway consists of a series of cytoplasmic steps, catalysed by ATP-dependent ligase enzymes, followed by a series of steps involving lipid-linked intermediates. TDHB’s group, in collaboration with Prof. C. Dowson and Dr. D. Roper (Biological Sciences), has considerable experience of studying enzymes on this pathway [2], and we are able to produce the intermediates and enzymes for all the cytoplasmic steps, and the lipid-linked cycle.


Project timing1




MurNAcL-AlaD-GluL-LysD-AlaD-Ala

PP


GlcNAc

PP


GlcNAc

(aa)n

PP


GlcNAc

(aa)n


GlcNAc

(aa)n

PP


GlcNAc

(aa)n

PP

P

PP

P

MraY

MurG

UDPGlcNAc

Fem ligases

aa-tRNA

trans-glycosylase

UDP-MurNAc-pentapeptide

UMP

peptidoglycancross-linking

CYTOPLASM

CELL SURFACE

UDP-MurNAc-L-Ala-D-Glu-L-Lys

D-Ala-D-AlaATP

D-AlaDdlB

ATPMurF

MurEL-LysATP

MurDD-GluATP

MurCL-AlaATP

UDPMurNAcMurB

NADPHFADH2

MurAPEP

UDPGlcNAc

flippase?

OH

CYTOPLASMICMEMBRANE

trans-peptidase

PS15

We have recently developed a new method to label the pathway intermediates with fluorescent groups, by insertion of an unnatural D-Cys residue at position 4 of the pentapeptide chain, in place of D-Ala [3]. A recent paper in Angewandte Chemie has reported that it is possible to label mammalian cell membranes using fluorescent phospholipid analogues applied externally [4]. This mini-project will explore whether it is possible to label the cell membranes of Escherichia coli or Bacillus subtilis using fluorescently-labelled lipid intermediate II. If so, then it will be possible in future to image the formation of fluorescent peptidoglycan in whole cells, using confocal microscopy. B. Mini-project Programme. Peptidoglycan precursor UDPMurNAc-pentapeptide containing D-Cys at position 4 of the peptide chain will be prepared from dipeptide D-Cys-D-Ala, using enzymatic methods developed in the group [3], and the D-Cys thiol will be labeled with fluorescein or rhodamine fluorophores. This material will then be converted into fluorescent lipid II, using M. flavus membranes, a procedure used currently in the group. The fluorescent lipid II will then be suspended as vesicles, and applied to growing E. coli or B. subtilis cells, washed, and the cells analysed by confocal microscopy (training will be provided by members of Prof. P. Unwin’s group in Chemistry). Derivatives in which the diphosphate linker is protected as a trimethylsilyl ether will also be tested, in case these are taken up more efficiently by cells. If successful, confocal microscopy will be used to monitor changes in fluorescence during cell division. C. Skills: The project will involve preparation & purification of fluorescent materials, basic microbiology, use of fluorescence spectroscopy, and confocal microscopy. D. Consumables budget: £300 for UDPMurNAc-pentapeptide, undecaprenyl phosphate, fluorescence reagents. E. References [1] “Bacterial Peptidoglycan Biosynthesis and its Inhibition”, T.D.H. Bugg, in “Comprehensive Natural Products Chemistry”, (ed. M. Pinto), Vol. 3, pp. 241-294, Elsevier, 1999. [2] P.E. Brandish, M. Burnham, J.T. Lonsdale, R. Southgate, M. Inukai, and T.D.H. Bugg, J. Biol. Chem., 271, 7609 (1996); N.I. Howard and T.D.H. Bugg, Bio-Org. Med. Chem., 11, 3083 (2003); J.J. Li and T.D.H. Bugg, Chem. Commun., 182 (2004); A.J. Lloyd, P.E. Brandish, A.M. Gilbey, and T.D.H. Bugg, J. Bacteriol., 186, 1747 (2004). [3] J.A. Schouten, S. Bagga, A.J. Lloyd, G. De Pascale, C.G. Dowson, D.I. Roper, and T.D.H. Bugg, Molecular Biosystems, 2, 484-491 (2006). [4] A.B. Neef & C. Schultz, Angew. Chem., 48, 1498 (2009).



Project title: Antibiotic peptides and their interactions with lipid membranes _________________________


Name: Matthew Hicks _____________________

Department: Chemistry _________________________ Building, Room: Chemistry B603__________

E-mail address: [email protected] _________ Phone number: ext 23293 ______________



Name: Alison Rodger _____________________________

Department: MOAC _______________________________ Building, Room: MOAC director’s office _____

E-mail address: [email protected] ______________ Phone number: ext 74696 _______________

Project outline:

A. Background to projectMany bacteria are becoming resistant to the antibiotic drugs that we have available at present.This is a major problem resulting in many deaths and it is of particular concern in hospitalswhere infections such as MRSA cause many complications. One class of antibiotic compoundthat is found in all types of organism are antibiotic peptides. These work via a variety ofmechanisms and target different parts of the bacteria. One type of antibiotic peptide works bymaking holes in the bacterial membrane and causing the ions inside the bacteria to leak outresulting in the death of the bacteria. An extremely important aspect of this action is thespecificity with which the peptide works: it’s no good having a peptide that effectively killsbacteria if it also kills the cells of the organism producing the peptide! However, the mechanismby which specificity is conferred is poorly understood. If we can unravel these mechanisms itmay be possible to use this information to design compounds that are more effective at killingbacteria and less toxic to humans.The key to understanding these processes is to study the way in which peptides interact withlipid membranes and to identify the features of the peptide and the membrane that mediate theinteractions. At Warwick we have been developing UV-visible linear dichroism (LD) in concertwith other techniques such as circular dichroism (CD), dynamic light scattering (DLS),fluorescence and absorbance, to probe the structure and kinetics of insertion of peptides andproteins interacting with model membranes: liposomes.1-5 Membrane protein systems areextremely important but very difficult to characterize and our facilities are unique in the world forthis purpose. In addition to this we collaborate with a group in Nottingham who have developeda computer program that will generate LD and CD spectra from proposed structures. 6 This hasenabled us to model the orientation of the peptides in the membrane and compare this withexperimental measurements to help in elucidation of complex mechanisms.









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B. Programme of workThere are a group of antibiotic peptides found on the skin of frogs (to protect them frominfections) called aureins. These and other peptides will be the subject of this project. The aim isto measure spectroscopic data from the peptides in the presence of different membranes and todetermine the mode of binding of these peptides to the membrane. This will be carried out usingspectroscopic techniques in combination with computer modelling. The work will be dividedapproximately into:

One third modelling and calculation of spectra from the models.Two thirds for data collection of experimental samples.

Time allowing there may also be a component of sequence analysis of the different peptides toidentify common sequence motifs within this class of peptides. The work will be carried out inthe chemistry department in the biophysical chemistry lab. This laboratory has pioneered theuse of LD in the determination of membrane peptide orientation and it is likely that this work willconstitute part of a publication on which the successful student will be an author.

C. Skills to be learnedThe student will learn a variety of biophysical techniques including absorbance, fluorescenceCD, LD and DLS. This will consist of both skills in measurement of data and its interpretation.Building of computer models of peptide structures will be a key part of this project and thesestructures will be used to generate theoretical spectra using a program developed by the Hirstgroup in Nottingham. 6 The student will be expected to participate in group meetings at whichthey will present their work to other members of the group, this will improve the key skills ofcommunication and discussion of scientific research.

D. Resources required (note total mini-project budget per student is £450 unless a specialcase is made)

This project will cost approximately £200. This is for reagents (lipids and peptides).

E. Outline of a literature review, including starting references.

Here are some key references used in this proposal, for further discussion and referencesplease contact Dr Hicks by email.

1) Dafforn, T. R.; Rodger, A. COSB 2004, 14, 541;2) Rodger, A.; Rajendra, J.; Marrington, R.; Mortimer, R.; Andrews, T.; Hirst, J. B.; Gilbert, A. T.

B.; Halsall, D.; Dafforn, T.; Ardhammar, M.; Nordén, B.; Woolhead, C. A.; Robinson, C.;Pinheiro, T.; J., K.; Seymour, M.; Perez, N.; Hannon, M. J. In Biophysical Chemistry:Membranes and Proteins, ; Templer, R. H., Leatherbarrow, R., Eds.; The Royal Society ofChemistry: Cambridge, 2002, 3;

3) Hicks, M.R.; Damianoglou, A.; Rodger, A.; Dafforn, T.R.; “Folding and membrane insertion ofthe pore-forming peptide gramicidin occurs as a concerted process“ Journal of MolecularBiology, 2008, 383, 358-366

4) Rajendra, J.; Damianoglou, A.; Hicks, M.; Booth, P.; Rodger, P. M.; Rodger, A. Chem. Phys.2006, 326, 210;

5) Ennaceur, S.M.; Hicks, M.R.; Pridmore, C.J.; Dafforn, T.R.; Rodger, A.; Sanderson, J.M.“Peptide Adsorption to Lipid Bilayers: Slow Processes Revealed by Linear DichroismSpectroscopy” Biophysical Journal, 2009, 96

6) http://comp.chem.nottingham.ac.uk/dichrocalc/


Project title: Development of Solid-State NMR as a Biophysical Probe _____________________________

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Dr. Steven P. Brown

Department: Physics ___________________________ Building, Room: Millburn House, F10

E-mail address: [email protected] ____________ Phone number: (024 765) 74359 ______________

Project outline:

Solid-state NMR has much potential as an atomic-level probe of structure and dynamics in biological solids that are difficult to characterise by established techniques such as crystal diffraction and solution-state NMR, e.g., amyloid fibrils [1] and membrane proteins [2].

This project concerns the development and refinement of solid-state NMR pulse sequences [3] that are used in such biological applications to determine internuclear distances (typically up to 5Å) by probing dipolar couplings. Specifically, solid-state NMR experiments (go.warwick.ac.uk/nmr) will be carried out on model compounds (labelled amino acids), with the results being analysed with the help of computer programs (e.g. SPINEVOLUTION [4]) that simulate the spin dynamics for a particular NMR pulse sequence for up to 11 coupled nuclear spins. Consumables budget: £ 150 for consumables (solid-state NMR). References: [1] C. Wasmer, A. Lange, H. Van Melckebeke, A. B. Siemer, R. Riek, B. H. Meier, Science 2008, 319, 1523. [2] C. Ader, R. Schneider, S. Hornig, P. Velisetty, E. M. Wilson, A. Lange, K. Giller, I. Ohmert, M. F. Martin-Eauclaire, D. Trauner, S. Becker, O. Pongs, M. Baldus, Nature Structural & Molecular Biology 2008, 15, 605. [3] S. P. Brown, & L. Emsley. "Solid-State NMR", in Handbook of Spectroscopy, Vo-Dinh and Gauglitz (eds), Wiley (2003). [4] M. Veshtort, R. G. Griffin, J. Magn. Reson. 2006, 178, 248.






(8 weeks) X Biophysical chemistry Slot 2 (18/05 to 10/07/08) Submission of thesis by: 8.59 am 13/07/08; viva to be scheduled in agreement with supervisor


PS18

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Project title: Solid-State NMR of a Membrane Protein___________________________________________

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Dr. Steven P. Brown & Dr. Ann Dixon

Department: Physics & Chemistry _________________ Building, Room: Millburn House, F10; C503

E-mail address: [email protected] ____________ Phone number: (024 765) 74359 ______________

E-mail address: [email protected]_____________ Phone number: (024 761) 50037 ______________

Project outline:

Despite comprising over 30% of the proteins encoded in the human genome, and playing crucial roles in many cellular processes, transmembrane proteins are currently among the least well characterised substances in biology. This lack of information is a direct consequence of the natural environment of these proteins, in that they are embedded within the lipid bilayers that surround cells, and as such, extracting structural information using established crystal diffraction and solution-state NMR methods is challenging. Whereas over 30,000 water-soluble proteins have been successfully characterised to date, less than 100 membrane-associated structures are known, making this a prime area of research in order to develop a better understanding of their complex roles and functions. Solid-state Nuclear Magnetic Resonance techniques have the potential to be a site specific probe of these systems, for which experiments can be performed upon samples within hydrated lipid environments to reveal key structural parameters [1,2].

As part of an ongoing research program, protocols have been established for the embedding of membrane proteins (prepared by chemical synthesis with specific isotopically labelled sites) within hydrated lipid vesicles to give samples suitable for solid-state NMR analysis. In this project, it is planned to carry out solid-state NMR experiments on the membrane protein, Glycophorin A (GpA). GpA contains a single transmembrane domain (Fig. 1a) that inserts into membrane bilayers as a stable α-helix and drives protein dimerisation (Fig. 1b) [3]. One sequence motif that is critical for the correct folding of GpA is the Gly-x-x-x-Gly motif (or GG4 for short), where glycine residues located every four amino acids localise to the same face of the α-helix and pack tightly against the other helix (Fig. 1b) [4]. Solid-state magic-angle spinning (MAS) NMR experiments will be carried out (go.warwick.ac.uk/nmr) on GpA peptides incorporating selectively 13C labeled amino acids. The investigation will build on previous solid-state NMR studies of GpA carried out by Smith et al [5,6]. Specifically, two-dimensional 13C-13C MAS experiments will be employed whereby cross peaks are indicative of 13C-13C dipolar couplings will be employed so as to probe multiple carbon-carbon distances up to 5 Å.

Consumables budget†: £ 150 for consumables (solid-state NMR and sample preparation).








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References: [1] S. P. Brown, & L. Emsley. "Solid-State NMR", in Handbook of Spectroscopy, Vo-Dinh and Gauglitz (eds), Wiley (2003). [2] D. Huster, Prog. Nucl. Magn. Reson. Spectrosc. 46, 79 (2005). [3] M.A. Lemmon, J.M. Flanagan, J.F. Hunt, B.D. Adair, B.-J. Bormann, C.E. Dempsey, and D.M. Engelman, J. Biol. Chem., 267, 7683 (1992). [4] W.P. Russ, and D.M. Engelman, J. Mol. Biol., 296, 911 (2000). [5] S. O. Smith, D. Song, S. Shekar, M. Groesbeek, M. Ziliox, S. Aimoto, Biochemistry 40, 6553 (2001). [6] S. O. Smith, M. Eilers, D. Song, E. Crocker, W. W. Ying, M. Groesbeek, G. Metz, M. Ziliox, S. Aimoto, Biophys. J. 82, 2476 (2002).

† Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects


Project title: Structure-Function Analysis of the Carbohydrate Recognition Domain of the Human C-type

Lectin DC-SIGNR Receptor Via Solution NMR.

Supervisors (the person who will be doing the day to day supervision of the mini-project): 1. Name: Dan Mitchell_____________________________

Department: WMS __________________________ Building, Room: CSRI ____________________

E-mail address: [email protected] _______ Phone number: 02476 968596 _____________

2. Name: Ann Dixon ______________________________

Department: Chemistry ______________________ Building, Room: Physical Sciences, C503 ____

E-mail address: [email protected]_______ Phone number: 50037____________________

Supervisor’s advisor (for non-permanent members of staff or those on probation:

Name: Alison Rodger

Department: Chemistry ____________________________ Building, Room: Physical Sciences, B607 ____


Project outline: Background. Protein-carbohydrate interactions are essential for the healthy function of the mammalian innate immune system. Oligosaccharide-binding proteins of the C-type lectin family play major roles in host defense such as pathogen recognition, phagocytosis, cell adhesion and antigen processing. Structural analysis of C-type lectin binding to oligosaccharides has been extensively studied via x-ray crystallography, although real-time biophysical analysis via NMR is surprisingly sparse. The cell-surface receptor (DC-SIGNR; CD299) is a type II transmembrane protein expressed on liver and lymph node endothelium, placental blood vessels, and certain adult stem cells (1, 2). DC-SIGNR has been shown to interact with deadly viruses such as HIV and SARS, enhancing their infectivity (3, 4) and represents an attractive target for antiviral therapy. The C-terminus of DC-SIGNR consists of a C-type lectin carbohydrate-recognition domain (CRD, see Figure 1) that preferentially binds to oligomannose structures and has been successfully crystallised and resolved in complex with a high-mannose oligosaccharide, (5, 6). Although important ligand binding data have been obtained from these x-ray studies, detailed structural information for DC-SIGNR interactions with endogenous and viral oligosaccharide ligands remain to be described. The overall aim of this work is to probe the ligand binding characteristics and dynamic properties of DC-SIGNR in real time at temperatures within the physiological range. To that end, we have successfully generated and purified 15N-labelled DC-SIGNR CRD material to analyze using solution-state NMR methods. Preliminary 1H-15N ** Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. However, all marks must be submitted at the latest by Sep 21 2009, 12 noon.







Figure 1. Crystal structure of C-terminus of DC-SIGNR, composed of a C-type lectin carbohydrate-recognition domain

B1

heteronuclear single-quantum coherence (HSQC) spectra (see Figure 2) indicate good resolution and strong evidence of correct domain folding.

Programme of work. From this strong starting point, the student will begin to assign peaks in the NMR spectra to specific amino acid residues. This will be aided by the existence of a crystal structure, which can be used to predict the peaks in the HSQC spectrum. The student will also analyze the binding of small ligands (e.g. monosaccharides) to DC-SIGNR by titrating the ligand into the protein sample. After each addition, an HSQC spectrum will be acquired. The position of peaks corresponding to amino acids involved in ligand binding will shift significantly, and these shifts can be observed by overlaying consecutive HSQC spectra. If time permits, a similar approach will be applied to the analysis of larger ligand binding (e.g. oligosaccharides and glycopeptides). Students will learn the fundamental methods for protein structure determination using NMR, as well as sample preparation methods required for the technique. Resources required. £200 for NMR consumables (D2O, tubes, deuterated buffers) and consumables required for protein purification. Outline of literature review. • Function of DC-SIGNR. • Current knowledge of the structure and ligands for DC-SIGNR. • Solution-state NMR methods for protein structure determination. Starting references. 1. E. J. Soilleux, R. Barten, J. Trowsdale, J Immunol 165, 2937 (Sep 15, 2000). 2. Y. Chen et al., J Exp Med 204, 2529 (Oct 29, 2007). 3. A. A. Bashirova et al., J Exp Med 193, 671 (Mar 19, 2001). 4. S. A. Jeffers et al., Proc Natl Acad Sci U S A 101, 15748 (Nov 2, 2004). 5. D. A. Mitchell, A. J. Fadden, K. Drickamer, J Biol Chem 276, 28939 (Aug 3, 2001). 6. H. Feinberg, D. A. Mitchell, K. Drickamer, W. I. Weis, Science 294, 2163 (Dec 7, 2001).

Figure 2: 1H-15N HSQC spectrum of DC-SIGNR CRD material obtained in 20 mM phosphate buffer, pH 6.0. The peaks in HSQC are well-dispersed, indicating a well-folded protein sample, and do not suffer from extensive overlap.


Project title: Structure/function studies on ALDC, part 2; Crystallography

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Professor Vilmos Fulop

Department: Biological Sciences ___________________ Building, Room: Biol SCi , Room B128 ________

E-mail address: [email protected]________ Phone number: (024 765)72628

Supervisor’s advisor (for non-permanent members of staff or those on probation: academic who agrees to provide supervision support to the supervisor and also agrees to meet the student briefly at least once a week): n/a.

Name:__________________________________________ Department: ____________________________________ Building, Room: ________________________ E-mail address: __________________________________ Phone number: _________________________ Project outline: A Background: This mini-project proposal is the second of three directed towards the study of the mechanism of action of the enzyme ADLC. The objective of the first mini-project will be to prepare two substrate and transition state analogues for co-crystallization with the isolated enzyme (Professor Martin Wills). The second mini-project will be the X-ray crystal structure analysis of ALDC bound to the analogues (Professor V. Fulop). The third mini-project will involve a molecular modelling study of the enzyme, (Professor P. M. Rodger).

B Programme Acetolactate decarboxylase (ALDC) has the unique ability to decarboxylate both enantiomers of acetolactate to give a single enantiomer of the decarboxylation product, (R)-acetoin. The enzyme decarboxylates the normal substrate (S)- α-acetolactate (Scheme 1). It then catalyses tertiary ketol rearrangement of the (R)-enantiomer with the migration of the carboxylate group (Scheme 2). Because this degenerate rearrangement proceeds via a transition state with a syn arrangement of the oxygen functions, the product is (S)- α-acetolactate which is then decarboxylated in the normal way. The enzyme also catalyses the decarboxylation of (S)- α-acetohydroxybutyrate. (S)- α-Acetolactate and (S)- α-acetohydroxybutyrate, the products of decarboxylation of α-ketocarboxylates are the biosynthetic precursors of the amino acids valine and isoleucine .

Details of the overexpression, purification and crystallization of α-acetolactate decarboxylase are given in Najmudin et al. (2003). We recently solved the crystal structure of the enzyme from a SAD experiment to give a partial model to 2.3Å. The structure was completed using higher resolution data (2.0Å) in 3 different crystal forms and the resolution at the moment is being extended to 1.1Å. α-Acetolactate decarboxylase is a 2 domain α/β protein with no significant structural homology to any other protein. The N-terminus domain comprises a 7 β -strand mixed β-sheet, which is extended into second molecule of the dimer related by 2-fold symmetry to give a 14 β-strand β-sheet. The C-terminus domain is a β-cylinder comprising 5 anti-parallel β-strands and a long α-helix. It provides the three highly conserved histidines, which coordinate the metal ion (Zn2+). The coordination of the metal is completed by a conserved glutamate from the C-terminus and two water molecules. The likely catalytic site is completed by the highly conserved arginine, threonine and a further glutamate in the vicinity of the metal.





MOAC Mini Project X Experimental biology Slot 1 (19/3 to 15/05/09) Submission of poster by: 8.59 am 18/05/08; Talk at the annual conference

(8 weeks) Biophysical chemistry (essentially Chemistry) X Slot 2 (18/05 to 10/07/08)

Submission of thesis by: 8.59 am 13/07/08; viva to be scheduled in agreement with supervisor


B2

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The project shall require the preparation two substrate and transition state analogues for co-crystallization experiments. The synthetic chemistry, first part of the project, will be supervised by Professor M. Wills in the Department of Chemistry. The second mini-project (this one) will be aimed at the X-ray crystal structure analysis of analogues bound to the enzyme. The third mini-project will involve a molecular modelling study of the enzyme, in order to help elucidate its mechanism and will be supervised by Professor P. M. Rodger, Department of Chemistry. The three mini-projects thus provide a coherent series of studies with training in synthetic chemistry, enzyme crystallography and molecular modelling. It is anticipated that the student will go on to use this blend of skills in further independent research work. The project has potential to lead to a full PhD programme by systematic mutagenesis/kinetics/crystallography complemented by modeling substrate entry and turnover at the catalytic site using computational techniques.

C Skills In this section of this three part project: general protein crystallographic methods, including crystallization of enzyme-substrate/analogue complexes, X-ray data collection, model building and refinement will be employed with full training provided. We have sufficient amount of pure enzyme available already. We have obtained crystals in three different forms (using different chemical conditions resulting in different crystal packing of enzyme molecules), therefore it is highly likely that at least one of these forms will yield the appropriate complexes we are seeking for.

D. Consumables budget: £ 200 for reagents. E. Reference. Najmudin, S., Andersen, J.T., Patkar, S.A., Borchert, T.V., Crout, D.H.G. & Fülöp, V. (2003). Purification, crystallisation and preliminary X-ray crystallographic studies on acetolactate decarboxylase. Acta Cryst. D59, 1073-1075.


Project title: Hormone binding analysis of a putative ABA receptor protein

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Richard Napier (with Andrew Marsh in Chemistry [email protected], ext 24565 and Martin Sergeant in

Warwick HRI)



Project outline: Background: The hormone abscisic acid (ABA) regulates both germination and water balance in plants and the identification of the receptor for ABA is a prime target for developmental biologists. In 2006 two receptors for ABA were published (Razem et al, Nature 2006, 439:290; Shen et al, Nature 2006, 443:823) with a further one in 2007 (Liu et al, Science 2007, 315:1712). All three publications identified different proteins. All three have since been withdrawn or seriously doubted. The Warwick labs of Marsh and Napier developed Magic Tag® technology in order to identify novel binding partners (Dilly et al, Chemical Communications 2007, 27:2808). Also the basis of a successful spin-out company, the technique was combined with protein library display on viral particles. Using ABA as an immobilized ligand we selected putative ABA-binding proteins from the library, one in particular appeared especially attractive on the basis of bioinformatics work. Several hits came through secondary screening and two have been cloned into bacterial expression vectors and shown to over-produce soluble protein (M Sergeant, A Thompson unpublished). The Project will use these cell lines to express the proteins from the viral screens. The proteins will be purified using FPLC by means of an epitope tag inserted during cloning. Using negative and positive controls from the lab, you will use different activity assays to test whether or not the proteins bind free ABA. This will be the next test for the hypothesis that our screen identified a putative, novel ABA receptor. One set of assays will be radioligand binding assays, by precipitation and/or by equilibrium dialysis. The other will be to use either surface plasmon resonance (Biacore SPR) to test for binding to a well characterized biotinylated ABA conjugate, or isothermal calorimetry (ITC) (Risk et al. Nature 456, 11 Dec, E5) to test for binding to free ABA in solution. The method chosen will depend on the quantity of protein purified. All the binding data will be compared to the kinetics of the positive control, a cell line expressing a monoclonal antibody to ABA that we have characterized extensively. Skills to be learnt: protein expression, protein purification, small molecule binding assays, binding kinetics, affinity measurement, thermodynamics Costs: No more than £450, to include reagents, column use, Biacore chip







Mathematics/computing possibly Slot 3 (13/07 to 04/09/08)

Submission of paper by: 8.59 am 7/09/08 Talk: 3/10/08, MOAC Seminar Room

B3 and PS10


Project title: Part 1: Culture and purification of fusion antibody Part 2: Training data collection Part 3: Mathematical modelling and data analysis

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Richard Napier (with Sarah Lee (Warwick HRI) and Hugo van den Berg



Project outline: Background: We have developed a novel biosensor platform capable of measuring in real-time continuous changes in concentration of an aqueous analyte from living tissue, bioprocesses etc (patent applied for). The system is based on a Biacore surface plasmon resonance instrument, but adaptable to other sensor platforms. We use of the dissociation kinetic of analyte-specific antibodies. By making use of rate measurements, rather than end-point or equilibrium recordings, the instrument moves process recordings away from time-fractionated, point-based plots and on to continuous, time-resolved monitoring traces. It is label-free, works on microlitre sample volumes, has a broad dynamic range and uses microdialysis probes to sample directly from living tissue or bioprocesses (such as culture flasks or fermentors). To complement the data recording systems, Dr van den Berg has begun to develop data handling routines which convert changes in Resonance Units to analyte concentrations and to explore response time windows for these conversions. The projects are a set which start with the production of a well-characterized monoclonal antibody from a bacterial cell line and take you through all the steps of collecting calibration data and up to training and testing the graphical reconstruction algorithms use for biosensor output. The Projects: Project 1) We have used a single chain antibody fragment (scFv) extensively in our work. It binds abscisic acid (ABA, Mw 264) with high affinity and specificity. You will express it from a bacterial (E. coli) cell line, extract and purify it using affinity columns and FPLC. Our construct includes the scFv as a fusion protein with maltose-binding protein (MBP) as well as a polyHistidine tag giving MBP-scFv-His. It purifies readily on amylose-columns and then purification can be polished using size-exclusion or ion exchange chromatography. You will run gels and optical procedures to check on purity and concentration. You will then test for binding activity and quantitate active protein using the Biacore





MOAC Mini Project 1 Experimental biology Slot 1 (19/3 to 15/05/09) Submission of poster by: 8.59 am 18/05/08; Talk at the annual conference

(8 weeks) 2 Biophysical chemistry Slot 2 (18/05 to 10/07/08) Submission of thesis by: 8.59 am 13/07/08; viva to be scheduled in agreement with supervisor

3 Mathematics/computing Slot 3 (13/07 to 04/09/08) Submission of paper by: 8.59 am 7/09/08 Talk: 3/10/08, MOAC Seminar Room

Time-resolved biosensor development.B4

B4 PS11 T14

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(surface plasmon resonance (SPR) spectrometry). Time permitting you may extend Biacore analysis with some kinetic measurements. Skills to be learnt: protein expression, protein purification, small molecule binding assays, binding kinetics, affinity measurement, classic pharmacological characterizations. Costs: No more than £450 per project, to include reagents, column use, Biacore chip Project 2) Using your purified scFv you will use SPR to do a set of kinetic experiments. You will prepare a chip with three different ligand densities. The ligand is a biotinylated conjugate of ABA and this binds readily to streptavidin. In order to get different ligand densities you will coat a chip with different streptavidin densities. After testing chip regeneration conditions and the stability of the surface you will collect data at different scFv concentrations, evaluating it in BIAevaluate software. Having now established run conditions, you will saturate the chip with scFv and generate dissociation datasets, collecting curves in the presence of various concentrations of free ABA in the running buffer. The data will be analysed in BIAevaluate software and we will discuss the parameters needed for Project 3. Skills to be learnt: Small molecule binding assay, SPR technology with associated software, binding kinetics, affinity measurement, classic pharmacological characterizations. Costs: No more than £450 per project, to include reagents, column use, Biacore chip Project 3) You will now have datasets for kinetic dissociation of antibody from ligand in the presence of a range of free analyte concentrations. You will use the data for modelling and/or graphical reconstruction strategies to generate calibrations for the sensor system. These methods will be tested to identify new experiments needed to train the calibration model. Parameters such as flow rate might be explored, for example. You will then load the chip with a series of concentrations of analyte (free ABA) within one monitoring cycle and challenge the calibration model to determine these concentrations, assessing the time-resolution and accuracy of each change in order to improve the training envelope. If time permits, you will connect a microdialysis probe into the test flow and repeat the calibration and training datasets by repeatedly dipping the probe into solutions of analyte of different concentration or using a gradient pourer. Skills to be learnt: SPR spectrometry, kinetics, data handling in MATlab, graphical reconstruction methods, model training. Costs: No more than £450 per project, to include reagents, column use, Biacore chip Note: In Project 1 the labwork will be done at Warwick HRI. All SPR work, Projects 2 and 3 will be based in Biological Sciences and MOAC resp.

MOAC mini-project proposalSubmit up to TWO pages including this header page tohttp://www2.warwick.ac.uk/fac/sci/moac/intranet/miniproj/mp_proposal_submission_form_2008_9 by 9 February 2009. Anyqueries contact Mónica Lucena [email protected].


Project title: In vivo imaging of proteins that shape the plant ER


Name: Lorenzo Frigerio

Department: Biological Sciences_____________________ Building, Room: B122

E-mail address: [email protected] ______________ Phone number: 23181 _______________________

11 Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the

respective Course Director. However, all marks must be submitted at the latest by Sep 21 2009, 12 noon.

MOAC’s Annual conference is 27-30 May, 2009. Attendance is compulsory for students. They will present their first project during it. Supervisorsare very welcome.

Project suitable as: (tick all that apply) Project timing1:(tick all that apply)




http://www2.warwick.ac.uk/fac/sci/moac/intranet/miniproj/mp_proposal_submission_form_2008_9

mailto:[email protected]

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Project outline:

A. Background to the projectReticulons are a recently discovered family of eukaryotic membrane proteins. They are responsiblefor conferring curvature to the membrane of the endoplasmic reticulum (ER), by virtue of their unusualtopology (resembling a W within the lipid bilayer). This curvature generates the characteristicmorphology of ER tubules (Voeltz et al., 2006, 2007). Plants contain far more reticulon genes thanmammals (Oertle et al., 2003). This may reflect the need of plant cells to adapt the morphology oftheir ER in response to different anatomical, physiological and environmental constraints. Virtuallynothing is known about the function of reticulons in plants. We have performed the first functionalstudies on a plant reticulon isoform (Tolley et al. 2008). We have now cloned 18 of the 21 reticulongenes of Arabidopsis and fused their genomic sequences to the yellow fluorescent protein (YFP). Wehave generated transgenic plants.

B programme of workYou will screen a subset of these plantsby in vivo confocal microscopy and study the tissue specificityand intracellular localisation of specific reticulon isoforms. In particular, you will focus on the isoformswhich are likely to be involved in the gating of plasmodesmata and the isoforms which do not localiseto the ER membrane. You will also intitate the construction of TAP-tagged versions od selectedreticulon isoforms, as a first step towards the indeitifcation of reticulon interacting partners.

C. Skills to be learned1) In vivo and confocal microscopy2) basic molecular biology and plant transformation techniques.

D. resources requiredPlant growth media, restriction enzymes, microscopy consumables. £450

E. ReferencesOertle, T., Klinger, M., Stuermer, C. A. O. and Schwab, M. E. (2003) A reticular rhapsody: phylogenicevolution and nomenclature of the RTN/Nogo gene family. FASEB J. 17, 1238-1247.Tolley, N., Sparkes, I. A., Hunter, P. R., Craddock, C. P., Nuttall, J., Roberts, L. M., Hawes, C.,Pedrazzini, E. and Frigerio, L. (2008) Overexpression of a Plant Reticulon Remodels the Lumen ofthe Cortical Endoplasmic Reticulum but Does not Perturb Protein Transport. Traffic 9, 94-102.Voeltz, G. K. and Prinz, W. A. (2007) Sheets, ribbons and tubules - how organelles get their shape.Nat Rev Mol Cell Biol 8, 258-264.Voeltz, G. K., Prinz, W. A., Shibata, Y., Rist, J. M. and Rapoport, T. A. (2006) A Class of MembraneProteins Shaping the Tubular Endoplasmic Reticulum. Cell 124, 573-586.

TYPE OF PROJECT Wet SUPERVISOR’S NAME: Dr Phil Gould/ Professor A. J. Easton TITLE OF PROJECT Analysis of coupled translation in Mouse mRNAs BACKGROUND TO THE PROJECT Control of gene expression can be exerted at several levels. Of these, control of translation of mRNA is becoming recognised as a critical component in the pathway from gene to protein. Eukaryotic translation machinery is able to exploit a number of novel processes to express additional proteins from a single mRNA. Much of the understanding of these processes, such as capping, function of eukaryotic initiation factors (eIFs) and internal ribosome entry sites (IRES), have come from the study of viruses.

Members of the subfamily Pneumovirinae of the family Paramyxoviridae, given the collective name pneumoviruses, are responsible for acute respiratory infections in their hosts. Sequence analysis has shown that the M2 mRNAs of all pneumoviruses contain two open reading frames, conserved in location though not in sequence. The protein products from ORF-2 have been detected in virus infected cells. We have shown that ribosomes access the second ORF using a novel mechanism in which expression of the virus M2 ORF-2 protein is initiated at AUGs located upstream of the ORF-1 termination codon, and that expression from these initiation codons requires the prior termination of M2 ORF-1 translation in vivo.

An analysis of the human genome has identified in excess of 750 mRNAs which contain two overlapping ORFs organised in a similar ‘architecture’ to those in the M2 mRNA. From subsequent analysis of favourable candidates coupled translation has been shown for the first time in the human genome. Using the searching programme potential candidates will be selected from the mouse genome and analysed to ensure the universal use of this mechanism. AIMS OF THIS PROJECT 2 or more candidate mouse cellular transcripts will be chosen for study. Using mouse cDNA the first ORF will be cloned with a reporter gene in place of the second ORF. The plasmid will contain a bacteriophage T7 promoter to direct transcription of the genes encoded by the mRNA. The plasmid will be transfected and cellular extracts will then be assayed for evidence of expression of the reporter gene. A positive signal will indicate that the second ORF is being expressed. In unison a second, mutated form of the reporter construct will be generated in which the stop codon of the first ORF has been deleted, causing translation to terminate further downstream on the mRNA. The effect of this on expression of the second ORF will be determined to show whether the translation of the second ORF is coupled to translation of the first ORF. METHODS TO BE USED PCR will be to amplify one of the cellular genes of interest from cDNA or from a commercial clone if available. The second open reading frame of the gene will be fused in-frame with a reporter to allow expression to be monitored. The construct will be inserted into a plasmid by restriction enzyme digestion and ligation. Transformation into E. coli, isolation of the DNA and transfection into mammalian cell cultures will be carried out. Gene expression will be monitored using a standard assay system. REFERENCES Ahmadian, G., et al., Embo J, 2000. 19. 2681-9. Gould, P.S. and A.J. Easton, J Biol Chem, 2005. 280. 21972-80. Gould PS and Easton AJ J Virol. 2007 81. 8488-96.

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Project title: Using FRET to study the dynamics and biological function of PDI ____

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Robert Freedman __________________________

Department: Biological sciences _____________________ Building, Room: Biol.Sci B008 _____________

E-mail address: [email protected]___________ Phone number: ext 23516 ________________





Project outline: BACKGROUND

Protein disulphide isomerase (PDI) is the major folding catalyst and molecular chaperone involved in the formation of native disulphide bonds in proteins that enter the secretory pathway and are destined for secretion or insertion into the plasma membrane. It was the first ‘folding catalyst’ identified and its activity has been studied for many years. However we still know very little of its mechanism of action; specifically, we do not understand how it couples conformational change (folding) and chemical change (disulphide bond formation) in its protein substrates (1). We believe that the key to this is the mobility and dynamics of PDI and especially the relative motions of its different domains and linkers. The aim of this project is to probe the motions and flexibility of PDI in order to understand its mechanism of action.

PDI is structured as follows: (N-terminus) a-b-b’-x-a’-c (C-terminus) where a, a’, b and b’ are all protein domains of approximately 100 residues with similar tertiary structures known as the thioredoxin-fold (trx-fold), and a,a’ both contain active sites capable of catalysing thiol:disulphide interchange reactions, whereas b and b’ do not contain such sites but the b’ domain contains a non-specific site for binding unfolded regions of polypeptides (folding substrates). In addition, x is a short linker region (approx 20 residues) and c is a C-terminal extension with no role in protein folding. There is no full-x-ray structure of PDI from humans or other higher eukaryotes although structures of the individual domains are known. The inability to generate good crystals for x-ray diffraction implies that there is some flexibility in the molecule, with domains moving relative to each other. We have recently shown that the b’ domain is intrinsically flexible and that the x region can occupy different conformations (2). We can interpret these motions by reference to the structures of yeast PDI (pdi1p)





MOAC Mini Project X Experimental biology Submission of poster by: 8.59 am 18/05/08; Talk at the annual conference


x Slot 3 (13/07 to 04/09/08) Submission of paper by: 8.59 am 7/09/08 Talk: 3/10/08, MOAC Seminar Room

B9

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which have been solved by x-ray crystallography (3); these data define possible structures but without indicating the full range of motions. AIMS OF THE PROJECT We aim to study PDI mobility by FRET using extrinsic probes, non-protein fluorescent groups that can be attached as labels to specific sites. The technique measures the efficiency of energy-transfer between groups and since this efficiency is distance-dependent, it can be used to determine the average distance between specific sites. We will use a pair of such groups and couple one group to the reactive active-site of the a domain and a different group to the corresponding site on the a’ domain. The extent of fluorescence energy transfer (FRET) between these groups provides a measure of the distance between the sites. We will determine this average distance in i) wild-type PDI, ii) various mutant forms of PDI which restrict its motions and iii) in wild-type and mutant PDIs in the presence of small (peptide) and large (unfolded protein) ligands. This will enable us to determine how the a and a’ domains move relative to each other in solution in response to ligand binding, and hence to picture these motions as they occur in the enzyme’s functional cycle. TECHNIQUES TO BE USED AND SKILLS TO BE LEARNED Expression (in E.coli) and purification of PDI and mutants; selection of appropriate pair of fluorescent labels for efficient Forster transfer; optimization of reaction conditions to generate appropriate level of labeling; purification of labeled protein; determination of site and extent of labeling by mass spectrometry; basic steady-state fluorescence characterization of labeled products. RESOURCES Basic equipment for bacterial growth and protein purification; commercial fluorescent labels; good steady-state spectrofluorimeter; access to mass spectrometry REFERNCES

1. Protein disulfide isomerases exploit synergy between catalytic and specific binding domains Freedman, R.B. et al. (2002) EMBO Reports 3 136-140 2. Alternative conformations of the x region of human protein disulphide-isomerase modulate exposure of the substrate-binding b’ domain Nguyen, V.D., et al. (2008) J.Mol.Biol. 383 1144-1155 3. The crystal structure of Yeast PDI suggests co-operativity between its active sites Tian, G., et al. (2006) Cell 124 61-73

MOAC mini-project proposal Submit up to TWO pages including this header page to http://www2.warwick.ac.uk/fac/sci/moac/intranet/miniproj/mp_proposal_submission_form_2008_9 by 9 February 2009. Any queries contact Mónica Lucena [email protected]. Project title: Constructing mutations in the b’ and x regions of human PDI

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Robert Freedman __________________________

Department: Biological Sciences ____________________ Building, Room: Biol.Sci B008 _____________

E-mail address: [email protected]___________ Phone number: ext. 23516________________





Project outline: BACKGROUND Protein disulphide isomerase (PDI) is the major catalyst of the formation of ‘correct’ (native) disulphide bonds in secreted and cell surface proteins that pass through -- and fold within -- the ER lumen (1). There is still much that is unknown about how this enzyme and chaperone acts on the newly-synthesized proteins that are its substrates (2). The molecule comprises a series of domains and linkers and its overall organization is a-b-b’x-a’-c, where a and a’ are catalytically active thioredoxin-like (trx) domains, b and b’ are inactive trx domains, x is a linker and c is an acidic tail. Working with fragments of PDI (domains or pairs of domains) is a useful strategy for understanding how the complete protein works. We have recently shown that the x linker can have alternative conformations (3) either masking, or not masking the major ligand binding site located on the b’ domain. This could control access of substrates to the binding site. Certain mutations can affect how readily the b’ and x domains interact (3). AIMS OF THIS PROJECT The mutations that affect how readily b’ and x domains interact have, until now, been constructed and expressed either in full-length PDI or in a simple b’x construct. Neither of these is optimal for analysing their effects on ligand-binding and enzymic activity. For these analyses, it would be useful to construct these mutations in the bb’x domain pair or in the b’xa’c domain pair since these are the simplest proteins in which one can study, respectively, binding of misfolded protein substrates and enzymic activity. The project combines molecular biology and protein expression with the aim of constructing expression plasmids encoding for these mutant domains and expressing them in E.coli to provide samples of the mutant proteins. It is unlikely that there will be time within the project to characterise the expressed mutant proteins, although that would be desirable.. METHODS TO BE USED AND SKILLS TO BE LEARNED ** Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. However, all marks must be submitted at the latest by Sep 21 2009, 12 noon.




MOAC Mini Project x Experimental biology Submission of poster by: 8.59 am 18/05/08; Talk at the annual conference

(8 weeks) X Slot 2 (18/05 to 10/07/08) Submission of thesis by: 8.59 am 13/07/08; viva to be scheduled in agreement with supervisor

X Slot 3 (13/07 to 04/09/08) Submission of paper by: 8.59 am 7/09/08 Talk: 3/10/08, MOAC Seminar Room

B10

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Basic molecular biological manipulation of plasmids in E.coli; oligonucleotide-directed site-specific mutagenesis; gel electrophoresis for analysis of DNA; bacterial growth and inducer-dependent expression of target protein; SDS-PAGE analysis to monitor and optimize protein expression; protein purification by affinity and ion-exchhange chromatography.. RESOURCES REQUIRED Oligonucleotides for PCR mutagenesis; basic molecular biological reagents; basic equipment for molecular biology, microbial growth and protein purification. REFERENCES 1. Protein disulfide isomerases exploit synergy between catalytic and specific binding domains Freedman, R.B. et al. (2002) EMBO Reports 3 136-140 2. Substrate recognition by the protein disulfide isomerases Hatahet, F. & Ruddock, L.W. (2007) FEBS. J. 274 5223-5234 3. Alternative conformations of the x region of human protein disulphide-isomerase modulate exposure of the substrate-binding b’ domain Nguyen, V.D., et al. (2008) J.Mol.Biol. 383 1144-1155

Project no. (to be inserted by MOAC)

ATP

adenosine

20 s 50 pA


Project title: Exocytosis of adenosine in the brain?

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Mark Wall _________________________________

Department: Biological Sciences _____________________ Building, Room: b006 ____________________

E-mail address: [email protected] _____________ Phone number: 573772___________________


support to the supervisor and also agrees to meet the student briefly at least once a week): Name: _________________________________________



Project outline: The signaling molecule adenosine is involved in many crucial brain processes such as sleep, respiration and locomotion. It also has an important protective role during periods of oxygen starvation. However, it is still unclear how adenosine is released into the extracellular space. Previous studies have shown that it can be released following extracellular ATP breakdown or by transport from the cytoplasm. We have strong evidence that adenosine can be released by vesicular exocytosis, in a similar fashion to other neurotransmitters. This would be a very exciting discovery as it would directly link neural activity to adenosine release.





MOAC Mini Project X Experimental biology X Slot 1 (19/3 to 15/05/09) Submission of poster by: 8.59 am 18/05/08; Talk at the annual conference



Figure 1. Cerebellar adenosine release with no detectable ATP. Traces from adenosine and ATP biosensors in a cerebellar slice. An electrical stimulus at the arrow produces a signal on the adenosine biosensor but no signal on the ATP sensor. There is ~ 0.5 µM adenosine detected. The limit of ATP detection is ~ 75 nM.

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Using novel microelectrode biosensors, we have investigated mechanisms of adenosine release in the cerebellum and shown (Wall and Dale, 2007) that adenosine can be released by short trains of neural activity. This adenosine release is both Ca2+ and action potential dependent and is not blocked by agents which interfere with adenosine transport. As we are unable to measure any ATP release, we hypothesise that adenosine is directly released by exocytosis. However because ATP breakdown can be extremely rapid it is possible that we cannot measure ATP because it is broken down before detection. To address this last point, this project aims to test exactly how quickly ATP is broken down in the cerebellum in vitro. The student will measure the diffusion and breakdown of ATP in cerebellar tissue using novel ATP and adenosine biosensors with Dr Wall. Dr Richardson will construct a model will to study these processes. This will allow us to test whether it is possible for the observed adenosine signals to arise without the measurement of any ATP This work is part of a significant MRC grant between Wall and Dale in Biological Sciences and Richardson in Systems Biology. The student will join an active team working on the project and will contribute to a publication. The project will provide training in biological experimentation.

Project title: PERCEPTION OF PAIN: THE EFFECTS OF UPREGULATION OF ENDOCANNABINOIDS ON DORSAL ROOT AFFERENT-MEDIATED SYNAPTIC TRANSMISSION

Supervisor Name: Dave Spanswick

Department: Warwick Medical School ___________________Building, Room: A012 Gibbet Hill Campus _______

E-mail address: [email protected] ___________ Phone number: 74868 ___________________

Project outline:

A) GENERAL BACKGROUND Endocannabinoids are lipid mediators with putative protective effects in many diseases of the nervous system. Recent studies undertaken in this laboratory indicate modulation of the endocannabinoid system in a rodent model of neuropathic pain. Increases in basal levels of the endocannabinoids anandamide and palmitoylethanolamide (PEA) follow induction of chronic pain, by peripheral nerve ligation, or spinal cord contusion. The synthesizing enzyme NAPE-phospholipase D is subject to upregulation and the degradative enzyme FAAH is downregulated. Electrophysiological studies of dorsal root afferent-mediated synaptic transmission to spinal dorsal horn lamina II neurones is subject to a tonic depression mediated through endocannabinoids, an effect enhanced following exposure to selective FAAH inhibitors and reversed by selective CB(1) receptor antagonists. The effects of these agents suggest a presynaptic role for CB receptors in modulating nociceptive afferent-mediated inputs. However, the extent to which the endocannabinoid system is functionally upregulated in neuropathic pain states and the role of CB(2) receptors, the latter also upregulated in neuropathic pain, remains unknown. Thus here we aim to investigate the effects of upregulation of the endocannabinoid anandamine, in neuropathic pain states, on dorsal root afferent-mediated synaptic transmission to spinal lamina II neurones and the functional role of CB1 and CB2 receptors in mediating the effects of endocannabinoids on nociceptive information processing.






Mathematics/computing x Slot 3 (14/07 to 05/09/08) Submission of paper: 8.59 am 8/09/08 Talk: 3/10/08, MOAC Seminar Room

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B) PROGRAMME OF WORK The aim of this miniproject is to investigate the role of endocannabinoids in modulating synaptic transmission in nociceptive information processing spinal cord dorsal horn neurons and the functional consequences of modulation of the system for nociceptive information processing following the establishment of a neuropathic pain state. The functional roles of CB1 and CB2 receptors in mediating the effects of endocannabinoids in spinal dorsal horn neurons will be determined. Experimental procedure Adult male Sprague-Dawley rats, weighing 200-230 g at the time of surgery, will be used. The baseline paw withdrawal threshold (PWT) will be examined using a series of graduated von Frey hairs (see below) for 3 consecutive days before surgery and re-assessed on the 7th to 8th and on the 12PthP to 14PthP day after surgery. Rodent models of neuropathic pain will be prepared as described by Kim and Chung (1992). Each rat will be anaesthetized with 5% isoflurane mixed with oxygen (2L per min) followed by an i.p. injection of sodium pentobarbitone at 50 mg/kg. In a prone position a para-medial incision will be made on the skin covering the L4-6 level. The L5 spinal nerve will be carefully isolated and tightly ligated with 6/0 silk suture. The wound will then be closed in layers after a complete hemostasis. A single dose of antibiotics (Amoxipen, 15 mg/rat, i.p.) will routinely be given for prevention of infection after surgery. The animals will be placed in a temperature-controlled recovery chamber until fully awake before being returned to their home cages. 10-14 days following surgery, Chung model rats will be terminally anaesthetised with isoflurane, the spinal column rapidly removed and 350–450 µm para-saggital slices cut using a Leica VT1000S vibrating microtome. Whole-cell patch clamp recordings will be undertaken in the L5 region of the spinal cord. A bipolar concentric stimulating electrode will be placed on the attached spinal dorsal root/DRG to stimulate postsynaptic currents in the recorded neuron. The effects of anandamide, selective FAAH inhibitors and CB1 and CB2 receptor anatagonists will be investigated on dorsal root afferent-mediated synaptic transmission in spinal lamina II neurones and on spontaneous miniature synaptic potentials recorded in these neurones under control conditions and following the establishment of a neuropathic pain state.

Ethical aspects The student will not work with live animals, only with tissue slices. However, the student will be asked to be present at the surgical procedure, in order to gain a thorough understanding of the scientific context of the project. The pain induced in the animal is equivalent to sciatica and is carefully monitored to ensure that it involves no more than mild discomfort; the procedure is tightly regulated. C) SKILLS TO BE LEARNED The project involves the use of whole-cell patch clamp electrophysiological recording techniques in isolated spinal cord slice preparations, neurophysiological and pharmacological techniques. The student will also have opportunity to gain experience of in vivo surgical techniques for the development of rodent models of disease and behavioural investigations. Basic single-cell molecular techniques may also be employed if the student expressed an interest in this area. D) RESOURCES REQUIRED None. E) STARTING REFERENCES Please contact Dave Spanswick.

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T r i t icum ae s t i v um(I / I I) ------MG--C---DDKCG---CAVP--CPGGTGCRCT 22

P ice ag l a uca ------MSGTC-TRGPACG---CGET--CACVD-CKCG 25Pseudo t sugamenz i e s i ------MASTC-TRGAECG---CGET--CACVD-CKCG 25La r i xkaempefe r i ------MASTC-TRGPECG---CGET--CACVD-CKCG 25P i n u st aeda ------MSS---TRSPQCG---CGET--CACAD-CKCG 23Chamaecypa r i sob t u s a ------MEGTCGCVATHCG---CLANGNCTCST-CRCA 28Cryptomer i aj apon ica ------MEGSCACVTSSCG---CLTNGNCTCDT-CKCA 28

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MOAC mini-project proposal Submit up to TWO pages including this header page to http://www2.warwick.ac.uk/fac/sci/moac/intranet/miniproj/mp_proposal_submission_form_2008_9 by 9 February 2009. Any queries contact Mónica Lucena [email protected]. Project title: Investigating the structural basis of metal binding and dynamics in metallothionein clusters – Part 1

Supervisor (the person who will be doing the day to day supervision of the mini-project): Name: Dr Oksana Leszczyszyn

Department: Chemistry ____________________________ Building, Room: Chemistry, A112



support to the supervisor and also agrees to meet the student briefly at least once a week): Name: Dr Claudia Blindauer

Department: Chemistry ____________________________ Building, Room: Chemistry, C510


Project outline: Background and Motivation. Metallothioneins (MTs) are small, cysteine-rich metalloproteins. These diverse proteins bind large amounts of both essential and non-essential metal ions such as Zn, Cu, Cd, Hg and As, in specialised metal-thiolate structures known as clusters. The most well-characterised MT domains are α and β clusters, which bind four and three metals respectively. The Blindauer group has recently elucidated the solution structure of a new cluster expressed in a MT from common wheat (see figure). This binuclear cluster will be the focus of this study.

Bioinformatic analysis has identified over 50 homologous binuclear domains in a diverse array of plant species. These homologues can be mapped through time from the relatively young seeding plants (first identified in the fossil record ca. 400 million years ago) to older evolutionary branches such as mosses and liverworts. In the majority of these plant species (i.e. monocots and dicots) the positions of the key cysteine metal binding residues are highly conserved. However, in tree species significant variations in both the number and position of cysteines have been observed. Given that the number and arrangement of cysteines in MT clusters has been shown to have important implications for structure, metal binding stoichiometry and dynamics, it is hoped that this

work would shed light on this phenomenon and begin to answer the question of what biochemical advantages or

differences such structural adaptations would confer. Aim. This is the first of two 8-week projects that fit into a wider research proposal which aims to investigate the effect that variations in cysteine number and position have on the stoichiometry, structure and metal binding dynamics of MT clusters. The objectives of this part of the study focus on generating clones of four binuclear






(8 weeks) Biophysical chemistry x Slot 2 (18/05 to 10/07/08) Submission of thesis by: 8.59 am 13/07/08; viva to be scheduled in agreement with supervisor


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clusters from different plant species: (i) Triticum aestivum, (ii) Picea glauca, (iii) Pinus taeda and (iv) Chamaecyparis obtusa, and developing a method for their purification. This initial work is fundamentally important.

Programme of work. The researcher will be required to generate four clones (as outlined above) using standard molecular biology techniques. The next stage will involve optimization of recombinant expression and the development of a robust purification method. Finally, identification and initial characterization of the protein products using a suite of biological and analytical techniques including ESI-MS and ICP-OES will be employed.

Skills to be learned. It is hoped that during this project you will develop wide ranging technical skills from molecular biology to protein purification, and including mass spectrometry of biological samples. Resources required. A small budget will be required for consumables involved in cloning (enzymes) and general protein purification. This is estimated at approximately £100-£150. Recommended reading. O.I. Leszczyszyn, R. Schmid, C.A. Blindauer, 2007, Proteins, 68(4): 922-935 O.I. Leszczyszyn, C.D. Evans, S.E. Keiper, C.A. Blindauer, 2007, Inorganica Chimica Acta, 360(1):3-13 M.J. Cismowski, S.S. Narula, I.M. Armitage, Journal of Biological Chemistry, 1991, 266(36):24390-24397 E. Freisinger, 2008, Dalton Transactions, 47: 6663-6675 M.L. Chernaik, P.C. Huang, 1991, PNAS, 88(8):3024-3028

Project title: THE EFFECTS AND MECHANSMS OF ACTION OF THE GUT-DERIVED HORMONE OXYNTOMODULIN ON NEURONAL EXCITABILITY OF HYPOTHALAMIC ARCUTE NUCLEUS NEURONES

Supervisor Name: Dave Spanswick

Department: Warwick Medical School ______________________ Building, Room: A012 Gibbet Hill Campus________

E-mail address: [email protected] ______________ Phone number: 74868____________________

Project outline:

A) General background Maintaining and co-ordinating energy balance and bodyweight is a function of specific areas of the brain which integrate information regarding the energy status of the body and formulate appropriate responses to maintain energy balance and bodyweight within narrow limits. Of the brain areas dedicated to controlling energy balance and bodyweight, certain regions of the hypothalamus, such as the arcuate nucleus, have emerged as key centres in the brain involved in sensing and formulating appropriate responses to changes in energy status. However, it remains largely unknown precisely how these brain areas function to detect changes in nutrient levels and energy stores signalled via chemical proteins called hormones and carried by the blood, and then produce appropriate changes in behaviour to counteract these changes in energy status. Interestingly, post-translational processing of glucagons in the intestine and the central nervous system yields a number of bioactive peptides, including oxyntomodulin (OXM). OXM is released from intestinal L cells into the blood in response to food ingestion in amounts proportional to caloric content; OXM potently suppresses appetite and reduces food intake in humans and is thought to mediate these anorexigenic effects by modulating neuronal activity in the arcuate nucleus (ARC) of the hypothalamus (1,2). A distinct receptor for OXM has yet to be identified, as are the cellular mechanisms by which this gut hormone regulates excitability of ARC neurones. Preliminary data collected in our lab suggest both pre-and post-synaptic mechanisms of action on ARC neurones.

B) PROGRAMME OF WORK Whole-cell electrophysiological recordings from neurons in the arcuate nucleus, in isolated rat brain slices, will be obtained using methods similar to those described in detail previously (3,4). The brain will be rapidly removed from adult rats and coronal 350µm slices containing the ARC prepared. Slices will be maintained at room temperature in



MOAC Mini Project √ Experimental biology x Slot 1 (26/3 to 16/05/08) Submission of poster: 8.59 am 19/05/08; Talk at the annual conference


Mathematics/computing x Slot 3 (14/07 to 05/09/08) Submission of paper: 8.59 am 8/09/08 Talk: 3/10/08, MOAC Seminar Room

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oxygenated artificial cerebrospinal fluid (aCSF) for at least an hour prior to recording. For recording, slices will be transferred to a custom-made recording chamber and continuously perfused at room temperature with aCSF of the following composition (in mM): 127.0 NaCl, 1.9 KCl, 1.2 KH2PO4, 26.0 NaHCO3, 10.0 D-glucose, 1.3 MgCl2, 2.4 CaCl2, equilibrated with 95% O2, 5% CO2, pH 7.3-7.4. Recordings will be obtained with patch pipettes filled with solution comprised of (mM): 140.0 Kgluconate, 10.0 Hepes, 10.0 KCl, 1.0 EGTA, 4.0 Na-ATP and had a pH of 7.4. Current and voltage data will be displayed on a digital oscilloscope (Gould DSO1602) and stored on DAT-tape (Biological DTR-1204, Intracel, Royston,UK) and as a digital file on the computer. For data analysis signals were digitized at 2-10kHz, stored and analyzed on a personal computer running pClamp8 software (Axon Instruments). The effects of OXM on orexigenic NPY/AgRP neurons and anaorexigenic POMC neurons will be tested using current and voltage-clamp recording techniques to identify pre- and post-synaptic sites and mechanisms of action of OXM. The specific programme of work will include:

1. Characterise postsynaptic ionic mechanisms underlying OXM-induced changes in electrical excitability of NPY/AgRP and POMC neurons. 2. Characterise presynaptic mechanisms of OXM-induced changes in neuronal activity using paired-pulse stimulation protocols together with analysis on miniature post-synaptic effects in the presence of TTX. 3. Charactersise the role of G-protein coupled receptors in mediating OXM-mediated effects using modulators of G-protein activity (GTPγs and GDPβs, etc).

The aim of this miniproject is to investigate the cellular mechanisms by which the gut-derived hormone oxyntomodulin acts at the level of the arcuate nucleus of the hypothalamus to regulate neuronal excitability, food intake and bodyweight.. C) SKILLS TO BE LEARNED The project involves the use of whole-cell patch clamp electrophysiological recording techniques in isolated brain slice preparations, neurophysiological and pharmacological techniques. Basic single-cell molecular techniques may also be employed if the student expressed an interest in this area. D) RESOURCES REQUIRED None. E) STARTING REFERENCES 1. ChaudhriOB, Parkinson JR, Kuo YT, Druce MR, Herlihy AH, Bell JD, Dhillo WS, Stanley SA, Ghatei MA, Bloom SR. Differential hypothalamic neuronal activation following peripheral injection of GLP-1 and oxyntomodulin in mice detected by manganese-enhanced magnetic resonance imaging. Biochem Biophys Res Commun 350: 298–306, 2006 2. DakinCL, Small CJ, Batterham RL, Neary NM, Cohen MA, Patterson M, Ghatei MA, Bloom SR. Peripheral oxyntomodulin reduces food intake and body weight gain in rats. Endocrinology 145: 2687–2695, 2004 3. Spanswick, D., Smith, M.A., Groppi, V.E., Logan, S.D. & Ashford, M.L. Leptin inhibits hypothalamic neurons by activation of ATP-sensitive potassium channels. Nature 390, 521-5 (1997). 4. van den Top, M., Lee, K., Whyment, A.D., Blanks, A.M. & Spanswick, D. Orexigen-sensitive NPY/AgRP pacemaker neurons in the hypothalamic arcuate nucleus. Nat Neurosci 7, 493-4 (2004).

Documents

MOAC MSc Miniproject Proposals Academic year: 2008 · Nicholas J Kruger and Antje von Schaewen (2003) The oxidative pentose phosphate pathway: structure and organisation. Current