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Page 1:  · ribosome. For the experiment, [2H,13C,15N, 100% HN]-labeled TF-RBD (13 kDa) complexed with unlabeled 50S (1.4 MDa) was sedimented in a 1.3 mm MAS rotor and 15N-1H correlation
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Page 3:  · ribosome. For the experiment, [2H,13C,15N, 100% HN]-labeled TF-RBD (13 kDa) complexed with unlabeled 50S (1.4 MDa) was sedimented in a 1.3 mm MAS rotor and 15N-1H correlation

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Page 4:  · ribosome. For the experiment, [2H,13C,15N, 100% HN]-labeled TF-RBD (13 kDa) complexed with unlabeled 50S (1.4 MDa) was sedimented in a 1.3 mm MAS rotor and 15N-1H correlation

3

14th

Chianti Workshop

Magnetic Resonance for Cellular Structural Biology

Book of Abstracts

5-10 June 2016, Principina Terra (Grosseto), Italy

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Page 6:  · ribosome. For the experiment, [2H,13C,15N, 100% HN]-labeled TF-RBD (13 kDa) complexed with unlabeled 50S (1.4 MDa) was sedimented in a 1.3 mm MAS rotor and 15N-1H correlation

5

Program Committee

Lucia Banci (University of Florence)

David Fushman (University of Maryland)

Daniella Goldfarb (Weizmann Institute of Science)

Claudio Luchinat (University of Florence)

Roberta Pierattelli (University of Florence), Co-Chair

Tatyana Polenova (University of Delaware), Co-Chair

Local Organizers

Linda Cerofolini

Marco Fragai

Moreno Lelli

Enrico Luchinat

Giacomo Parigi

Enrico Ravera

with the help of

the CERM PhD students!

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6

Organizing Institutions

CERM

University of Florence

Fondazione Luigi Sacconi

CIRMMP

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7

We wish to acknowledge the contribution of

Biophysical Society

EBSA - European Biophysical Societies’ Association

GIDRM - Gruppo Italiano Discussione Risonanze Magnetiche

ISMAR - International Society of Magnetic Resonance

IUPAB - International Union for Pure and Applied Biophysics

SCISB - Società Chimica Italiana / Chimica dei Sistemi Biologici

for providing grants to early stage researchers and

BIOCLASS

Bracco Imaging

Bruker BioSpin

CORTECNET

EurisoTop

F1000 Research

Giotto Biotech

Gruppo SAPIO

JEOL

Marc de Grazia Selections

pNMR

SARSTEDT

Silantes

Spectra2000

for further support

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9

Abstracts

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Page 12:  · ribosome. For the experiment, [2H,13C,15N, 100% HN]-labeled TF-RBD (13 kDa) complexed with unlabeled 50S (1.4 MDa) was sedimented in a 1.3 mm MAS rotor and 15N-1H correlation

11

Modulation of amyloid β peptide aggregation by metal

ions

Axel Abeleina,b

, Astrid Gräslunda, Jens Danielsson

a

a Department of Biochemistry and Biophys ics, Stockholm Univers i ty, Svante Arrhenius väg

16, 106 91 Stockholm. b

Department of Neurobio logy, Care Sc ienc es and Society (NVS), Div is ion of

Neuroger iatr ics , Karol inska Ins t i tutet , 14157 Huddinge, Stockholm . (axel .abele [email protected])

Metal ions, in particular zinc and copper ions, have emerged to play a key role in the

aggregation process of amyloid β (Aβ) peptide that is closely related to the pathogenesis of

Alzheimer’s disease. A detailed understanding of the underlying mechanistic process of

peptide-metal interactions, however, has been challenging to obtain. By applying a

combination of NMR techniques and fluorescence kinetics methods we have investigated

quantitatively the thermodynamic Aβ-metal binding features as well as how metal ions

modulate the nucleation mechanism of the aggregation process. Our results show that,

under near-physiological conditions, sub-stoichiometric amounts of Zn2+ effectively retard

the generation of amyloid fibrils. The inhibition process shows an exponential dependence

of aggregation half times on the Zn2+ concentration. A global kinetic profile analysis reveals

that in the absence of Zn2+ Aβ40 aggregation is driven by a monomer-dependent secondary

nucleation process in addition to fibril-end elongation. In presence of Zn2+, the elongation

rate is reduced, resulting in reduction of the aggregation rate, but not a complete inhibition of

amyloid formation. We show that Zn2+ transiently binds to the N-terminus of the monomeric

peptide. A thermodynamic analysis supports a model where the N-terminus is folded around

the Zn2+ ion, forming a marginally stable, short-lived folded Aβ40 species. This conformation

is highly dynamic and only a few percent of the peptide molecules adopt this structure at any

given time point. Our findings suggest that zinc ions effectively prevent Aβ40 monomers to

be involved in fibril elongation. In this conceptual framework we suggest that zinc adopts the

role of a minimal anti-aggregation chaperone for Aβ40.

References:

Abelein A, Gräslund A, Danielsson J. Proc Nat Acad Sci of the U S A, 112(17), 5407-12 (2015).

Presented by: Axel Abelein

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12

Design and testing of paramagnetic agents for functional

and molecular MRI studies

Silvio Aime Department of Molecular Biotechnology and Health Sc iences and Molecular Imaging

Center , Univers ity of Tor ino

The possibility of carrying out Functional and Molecular Imaging protocols by means of

MRI is very attractive for the superb anatomical resolution that is attainable by this

technique. However, MRI suffers from an intrinsic insensitivity (with respect to the competing

imaging modalities) that has to be overcome by designing suitable amplification procedures

involving the use of properly designed chemicals. This approach relies first on the

development of paramagnetic contrast agents endowed with an enhanced sensitivity and on

the identification of efficient routes of accumulation of the imaging probes at the sites of

interest. In this context much attention has been devoted to the design and use of self-

assembled systems based on amphiphilic molecules as well on the use of whole cells,

where the imaging reporters are represented by highly stable paramagnetic Gd(III)

complexes.

Besides relaxation agents much attention is currently devoted also to the use of

Paramagnetic Metal Complexes as CEST agents (CEST= Chemical Exchange Saturation

Transfer). In these systems one exploits the paramagnetic perturbation in terms of its effect

on the shift of the resonance of protons that are involved in a slow/intermediate exchange

with water. Upon applying a second rf field at the absorption frequency of the exchangeable

protons, a net saturation effect is detected on the water signal. These are frequency

encoding systems that allow multiple agents detection in the same anatomical region as well

as they offer the possibility of designing innovative responsive probes that report on specific

parameters of the microenvironment in which they distribute. To overcome sensitivity issues,

also for this class of agents, the use of Liposomes (LipoCEST) and RBCs (ErythroCEST)

appear to offer valuable solutions.

Presented by: Silvio Aime

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13

Structures of protein RNA complexes using NMR, EPR

and Mass-spectrometry

G. Masliaha, G. Dorn

a, A. Leitner

a, O. Duss

a, M. Yulikov

b, R.

Aebersolda, G. Jeschke

b, F. Allain

a a

Department of Bio logy, ETH Zur ich, 8093, Zur ich, Switzer land . (a l la [email protected] l.ethz.ch) b

Department of Chemistry and Appl ied Biosc iences, ETH Zur ich, 8093, Zur ich,

Switzer land.

Protein-RNA-complexes are central for the regulation of gene expression and thus crucial

for cellular function. While solving structures of protein-RNA complex with NMR has been

very successful owing to the low affinity of many RNA-protein interactions, the size limitation

of the complexes that could be tackled structurally solely by NMR has remained an issue.

Using EPR and more recently Mass-spectrometry (MS) in combination with NMR has

allowed us to investigate the structure of larger protein-RNA complexes. Recent structural

results between the RNA binding proteins RsmE (Duss et al, Nature 2014), TRBP, and PTB

bound to RNA will be reported.

Presented by: Frédéric Allain

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14

The encounter complex between cytochrome P450cam

and putidaredoxin characterized by minimum occurrence

of regions (minOR) analysis

Witold Andrałojća, Yoshitaka Hiruma

b, Wei-Min Liu

b, Claudio

Luchinata, Masaki Nojiri

c, Giacomo Parigi

a, Enrico Ravera

a, Marcellus

Ubbinkb

aCERM, Univers ity of Florence, v ia Luig i Sacconi 6, 50019 Sesto Fiorent ino, I taly ,

(andralo [email protected] f i . i t ) .

bLeiden Ins t i tute of Chemistry, Leiden University , Einste inweg 55, 2333 CC Leiden, The

Nether lands . cDepar tment of Chemistry , Osaka Univers i ty

cRIKEN SPr ing-8 Center, Japan.

The structure of the electron transfer active complex between cytochrome P450cam and

putidaredoxin has recently been determined by X-ray crystallography and paramagnetic

NMR. However, many of the measured paramagnetic relaxation enhancements (PREs)

cannot be accounted for by this state, suggesting the presence of a lowly populated

encounter complex. The exquisite sensitivity of PREs to the even most sparsely populated

states, as long as they are located close to the paramagnetic probe, gives a unique

opportunity to look into this elusive state for the current system. However, the problem of

ensemble recovery from averaged NMR observables is inherently ill-defined. To tackle this

limitation we have introduced the concepts of Maximum and minimum Occurrences of

conformational regions (MaxOR and minOR)1 as the safest way to achieve quantitative

information from averaged experimental data. The MaxOR (or minOR) of a region is defined

as the largest (or smallest) amount of time that the system can spend in a given part of

conformational space and still be compatible with the experimental observables. We have

exploited the concept of minOR to determine the conformational regions that have to be

sampled by the complex. By assuming the sparsity of the actual conformational ensemble

we were able to select the most likely sampled structures. The distribution of the plausible

minor states obtained in this way correlates well with the electrostatic potential map around

P450cam, suggesting that we are indeed dealing with an electrostatics-driven encounter

complex.

References:

1. Andrałojć W, Luchinat C, Parigi G, Ravera E. J. Phys. Chem. B, 118 (36), 10576–10587 (2014)

Presented by: Witold Andrałojć

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15

Segmental Isotope Labeling of the multidomain LA

protein for NMR structural study

Aikaterini Argyrioua, Christine von Schroetter

b, Constantinos

Stathopoulosc, Frédéric Allain

b, Georgios A. Spyroulias

a

a Department of Pharmacy, Univers i ty of Patras, GR -26504, Patras, Greece

(argyr [email protected] ) . b Ins t i tute of Molecular Bio logy and Biophy s ics, ETH Zür ich, CH-8093, Zür ich, Switzer land.

b Department of Biochemistry, School of Medic ine, Universi ty of Patras, GR -26504, Patras,

Greece.

The LA protein (Lupus Antigen) was first described as an autoantigen in patients suffering

from rheumatic diseased systematic lupus erythematosus and Sjogren’s syndrome. LA from

the lower eukaryote Dictiostelium discoideum (354 a.a.) has 4 distinct domains1; LA motif

(LAM), two RNA recognition motifs (RRM1 & RRM2) and a C-terminus region. So far the

structure of the full-length LA protein and the role of RRM2 remain elusive.

Application of high resolution NMR spectroscopy for the study of the full-length protein

has to overcome the size of the protein and spectra quality; both factors may complicate the

analysis of the data. Therefore, segmental isotopic labeling is applied for the NMR study of

the LA protein. In our study, each construct in fusion with an intein was expressed

separately in Escherichia coli, allowing different isotopic labeling schemes. EPL (Expressed

Protein Ligation) is based on the reaction where a C-terminal α-thioester of the first domain

(RRM1) reacts with an N-terminal cysteine residue of the second domain (RRM2), resulting

in the formation of a native peptide bond and takes place on chitin column. The 15N-TROSY

spectrum of {15N-RRM1}-{14N-RRM2} and {14N-RRM1}-{15N-RRM2} revealed that RRM1 and

RRM2 are well folded and match fairly well with reference spectra of 15N-RRM1 and 15N-

RRM2, respectively.

References:

1. Argyriou AI, et al. Biomol NMR Assign 9, 219-22 (2015); Biomol NMR Assign 9, 303-7 (2015).

Acknowledgments: We acknowledge partial support from “The FEDERATION of EUROPEAN BIOCHEMICAL

SOCIETIES (FEBS)” for a FEBS Short-Term Fellowship. The work was also supported by " EU FP7-REGPOT-

2011 “SEE-DRUG” (nr. 285950 to C.C. & G.S.).

Presented by: Aikaterini Argyriou

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16

Ribosomal complexes by 1H-detected ultra-fast MAS

solid-state sedNMR

Emeline Barbet-Massina, Eli van der Sluis

b, Chih-Ting Huang

c, Venita

Daebeld, Shang-Te D. Hsu

c, Roland Beckmann

b, Bernd Reif

a

a Department Chemie Festkörper-NMR-Spektroskopie, Technische Univers ität München,

L ichtenbergstrasse 4,85748, Garching, Germany. (emel ine.barbet [email protected]) b

Gene Center, Ludwig -Maximi l ians-Univers ität München, Munich, Germany. cInst i tu te of Bio logical Chemistry , Academia Sin ica , Taipei, Taiwan.

dBruker Biospin GmbH, Rheinstet ten, Germany.

We present first MAS solid-state NMR data on ribosomal complexes that employ proton

detection and ultra-fast MAS, which allow to investigate large asymetric complexes in which

only a small molar fraction is isotopically labeled1. In particular, we focus of the ribosome-

binding domain of Trigger Factor (TF-RBD) which interacts with the 50S subunit of the

ribosome. For the experiment, [2H,13C,15N, 100% HN]-labeled TF-RBD (13 kDa) complexed

with unlabeled 50S (1.4 MDa) was sedimented in a 1.3 mm MAS rotor and 15N-1H

correlation experiments were recorded of TF-RBD, in the complex and monomeric in

solution. Chemical shift mapping allowed us to identify residues for which resonances are

affected upon binding; these residues cluster in alpha-helix 2 and in the “TF-signature” motif,

in line with previous biochemical and crystallographic data2,3. This remarkable result from a

methodological point of view opens new perspectives for the study of structure and

dynamics of ribosomal protein and ribosome nascent chain complexes by solid-state NMR.

References: 1. Barbet-Massin E, Huang CT, Daebel V, Hsu ST, Reif B. Angew. Chem. Int. Ed. 54, 4367-4369 (2015)

2. Kramer G, Rauch T, Rist W, Vorderwulbecke S, Patzelt H, Schulze-Specking A, Ban N, Deuerling E, Bukau

B. Nature 419, 171-174 (2002)

3. Schlunzen F, Wilson DN, Tian P, Harms JM, McInnes SJ, Hansen HA, Albrecht R, Buerger J, Wilbanks SM,

Fucini P. Structure 13, 1685-1694 (2005)

Presented by: Emeline Barbet-Massin

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17

MicroRNA function through structural dynamics

Lorenzo Baronti, Katja Petzold Department of Medical Biochemistry and Biophys ics , Karol inska Inst i tutet , Scheeles väg 2,

17165, Stockholm, Sweden. ( lorenzo.baront [email protected])

Our work aims at elucidating the structure and dynamics of microRNAs (miRNAs) by

solution state NMR. miRNAs are small non-coding RNA molecules that are able to fine-tune

target messenger RNA (mRNA) translation. The key interaction of miRNAs machinery is the

base-paring pattern with target mRNAs, which is non-conserved, extensively imperfect and

often predicted to be highly dynamic2. In this work we focus on the human miRNA-34a in the

frame of the p53 pathway targeting mRNA SIRT13. Once extensively assigned miR-34a

resonances in its free state, we were able to study the melting profile by chemical-shift

perturbation and define a transition between a stem-loop structure and a single-stranded

conformation. The titration performed with the unlabeled target mSIRT1 revealed the binding

event to be governed by slow exchange, in agreement with the formation of a stable RNA

duplex. Altogether the slow-exchange and the extended size prevented from assigning the

complex. In order to overcome such limitation and study the system in greater detail, we

designed and completely assigned a model to mimic the central bulge of the miR-

34a/mSIRT1 duplex. We present the preliminary results of Relaxation Dispersion (R1ρ)

experiments4 recorded on this model in order to identify possible alternative states within the

miR-34a/mSIRT1 duplex that potentially controls the mRNA stability.

References:

1. Bartel DP. Cell 116, 281-297 (2004)

2. Nakanishi K, Weinberg DE, Bartel DP, Patel JD, Nature 486, 368-374 (2012)

3. Yamakuchi M, Lowenstein JC, Cell Cycle 8, 712-715 (2009).

4. Dethoff EA, Petzold K, Chugh J, Casiano-Negroni A, Al-Hashimi HM, Nature 491, 724-728 (2012)

Presented by: Lorenzo Baronti

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18

Heat & Cold vs. DMSO Unfolding of Antifungal Proteins.

Hidden conformers found by CEST-NMR and MD

calculations

Gyula Battaa, Ádám Fizil

a, Dorottya Hajdu

a, Zoltán Gáspári

b,

Florentine Marxc

a Research Group for Structura l Bio logy, Depar tment of Chemistry , Univers ity of Debrecen,

Egyetem tér 1, 4032,Debrecen, Hungary. ([email protected]) bPázmány Péter Cathol ic Univers ity , Faculty of Informat ion Technology

Práter u. 50A, 1083 Budapest, Hungary . c

Medical Univers ity of Innsbruck, Biocenter , Div is ion of Molecular Bio logy, Innra in 80 –82,

6020 Innsbruck,Austr ia.

The antifungal disulfide protein PAF was shown earlier1 as a

three-state folder upon temperature perturbation in between the -

8oC ... +71oC range. A detailed analysis of thermal unfolding yielded

the thermodynamic parameters of unfolding, and suggested that a

considerable amount of NMR invisible conformers may persist in

aqueos buffer even at the highest stability temperature.

Conventional Lipari-Szabó model-free method reports on a rock-

hard protein on ps-ns timescale. However, molecular dynamics

calculations combined with chemical shift data support the presence

of different conformational clusters, while CEST-NMR provided evidence on sparsely

populated (0.15%) "strange" conformers in slow exchange with the major, visible conformer.

Chemical "denaturation" experiments revealed that urea can unfold completely neither

PAF nor the PAFD19S variant. In contrast, DMSO is an efficient "chemical denaturant" that

induces two-state unfolding of both proteins allowing thereby the determination of unfolding

parameters.

References:

1. Fizil Á, Gáspári Z, Barna T, Marx F, Batta G. CHEMISTRY-A EUROPEAN JOURNAL 21, 5136-5144 (2015)

Acknowledgments: Hungarian Grant OTKA ANN 110821 to G.B. and Austrian Science Fund FWF P25894 to

F.M.

Presented by: Gyula Batta

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19

Monitoring site-selective reactivity towards cross linking

in α-synuclein

Jon Werner-Allena, Jung Ho Lee

a, Rodney Levine

a, Jinfa Ying

a,

Julien Rochea, Ad Bax

a

a Laboratory of Chemical Physics , Nat ional Inst i tu te of Diabetes and Digest iv e and

Kidney Diseases, Nat ional Inst i tu tes of Heal th, B ethesda, MD 20892. ([email protected])

Chemical cross linking of proteins is widely used to study physical proximity and/or

interaction between proteins in a cellular environment. Most cross linkers bridge Lys amino

groups, but the reactivity of different Lys residues is generally unknown. Cross linking

between synuclein molecules in a cellular environment yields bands on a gel that

correspond to a tetrameric species and has been interpreted as evidence that the protein

exists as a folded α-helical tetramer, rather than an intrinsically disordered protein inside the

cell. By using ultra-high resolution NMR spectroscopy, we show that it is possible to

quantitatively evaluate the reactivity towards cross linking of individual Lys residues, in the

absence and presence of lipid membranes. The latter are highly protective towards cross

linking, but in a manner that depends on the lipid type.

Natural cross linking of α-synuclein inside the cell is known to be highly toxic and has

been implicated in Parkinson’s disease. In particular, the dopamine breakdown product, 3,4-

dihydroxyphenylacetaldehyde (DOPAL), is known to be highly reactive towards cross

linking, but the chemical mechanism of this process is unknown. Using NMR and LC-MS,

we identified Lys residues as the target of DOPAL reactivity and determined the structure of

the first reaction product in the cross linking process, revealing a surprising dicatechol

pyrrole lysine product. This novel modification results from the addition of two DOPAL

molecules to the Lys sidechain amine through their aldehyde moieties and the formation of a

new carbon-carbon bond between their alkyl chains to generate a pyrrole ring, and is

detectable in reactions with near physiological concentrations of DOPAL. The

dicatecholpyrrole-lysine adduct is itself quite unstable, and rapidly reacts in a variety of ways

with other residues, thereby inducing covalent cross links.

Presented by: Ad Bax

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20

Dynamics of the intrinsically disordered N-terminal

domain of the Hendra virus phosphoprotein as unveiled

by NMR spectroscopy

Matilde Beltrandia,b,c

, Isabella Fellic, Sonia Longhi

a,b and Roberta

Pierattellic

a Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB) UMR 7257,

13288, Marseille, France. bCNRS, AFMB UMR 7257, 13288, Marseille, France.

cCERM and Department of Chemistry Ugo Schiff, University of Florence, Via Luigi Sacconi 6, Sesto Fiorentino,

50019 Florence, Italy. ([email protected]).

Hendra virus (HeV), a bio-safety level 4 pathogen, belongs to the Paramyxoviridae family.

The HeV genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid.

This latter is the substrate used by the polymerase complex, made up the L protein and its

co-factor the phosphoprotein (P), during both transcription and replication. The P protein

possesses a long N-terminal disordered domain (PNT), responsible for the interaction with

the unassembled form of the nucleoprotein, and a C-terminal domain (PCT) consisting of

alternating disordered and structured regions. Nuclear magnetic resonance spectroscopy

(NMR) and small angle X-ray scattering (SAXS) are particularly well adapted to study

flexible macromolecules in solution. In particular, these studies are gaining momentum for

characterizing the conformational behavior of intrinsically disordered proteins (IDPs), i.e.

proteins that lack highly populated uniform secondary and tertiary structure under

physiological conditions in the absence of a partner. We are currently investigating the

Hendra virus PNT domain by a hybrid approach that combines NMR, SAXS and structural

modeling. The results arising from these studies are expected to contribute to a better

understanding of the dynamic and conformational features of this disordered region.

References:

1. Communie G. et al, PLoS Pathog, 9(9), e1003631 (2013)

Presented by: Matilde Beltrandi

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21

Recent insights in polarization transfer ENDOR and

Overhauser DNP

Marina Bennati Electron Spin Resonance Spectroscopy, MPI for Biophysical Chemistry and Univers i ty of

Göt t ingen, Am Fassberg 11, 37077, Göt t ingen, Germany. ([email protected])

Polarization transfer from electron to nuclear spins is fundamental not only to enhance

sensitivity in NMR experiments but also in EPR/ENDOR techniques. ENDOR permits to

detect magnetic nuclei that are in the vicinity of paramagnetic centers and typically not

visible in an NMR experiment. Though the importance and versatility, conventional ENDOR

requires considerable signal averaging, usually at low temperatures, particularly when

detecting low-gamma nuclei. Recent systematic studies have established that ENDOR

suffers not only from experimental imperfections but also from nuclear spin saturation1. To

address the issue, we have proposed a polarization transfer scheme inspired form

experiments proposed for DNP and NMR2. It is based on cross polarization, where two

spins are concomitantly irradiated to match their energy splitting. For electron and nuclear

spins, the largely different gyromagnetic ratios can be compensated by the hyperfine

coupling and a proper choice of resonance offsets3. Performance and application potential

of this experiment in ENDOR and NMR are presented4.

Moreover, we have been also investigating Overhauser DNP mechanism in liquid

solution. Recent studies have been focused on exploring the DNP scalar mechanism, which

potentially offers a much more favourable field dependence than the dipolar-relaxation

based mechanism. Our experimental set up and recent results for DNP at W-band are

presented.

References:

1. Rizzato R, Bennati M. PCCP 16, 7681-7685 (2014)

2. Weis V, Bennati M, Rosay M, Griffin RG. J. Chem. Phys. 113, 6795-6802 (2000)

3. Rizzato R, Kaminker I, Vega S, Bennati M. Mol. Phys. 111, 2809-2823 (2013)

4. Rizzato R, Bennati M. ChemPhysChem. 16, 3769-3773 (2015)

Presented by: Marina Bennati

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22

Functional divergence of the PHDVC5HCH domain within

the NSD family

Andrea Berardi, Giacomo Quilici, Maria Angeles Corral Rodriguez,

Fernando Martin, Michela Ghitti, Giovanna Musco

Biomolecular NMR Laboratory, Division of Genetics and Cell Biology, IRCCS S. Raffaele Scientific Institute,

Milan, Italy ([email protected])

The NSD transcription protein members (NSD1, NSD2, NSD3) contain several chromatin-

related modules (SET , PWWP and PHD domains) and they are implicated in

developmental diseases and cancer, but their mechanisms of action is still unclear1. The

PHDV and C5HCH module of NSD1 and NSD3 fold as unique functional unit (PHDVC5HCH)

and share a high sequence identity (~60%)2, but they have a divergent role in the

recognition of modified histones and of the co-repressor factor Nizp1 by its C2HR module.

Our data reveal for PHDVC5HCHNSD1 the non-specific interaction with H3 histone tail and the

high affinity for C2HRNizp1 (Kd~4μM)2, conversely PHDVC5HCHNSD3 bind with a similar affinity

(Kd~300μM) in two different binding pocket both the H3K9me3 3 and C2HRNizp1. On NSD1

and NSD3 the C2HRNizp1 binding site localizes at the interface between the two PHD fingers

in a distinct region from the canonical histone binding site. Additionally, NMR titrations

indicate that H3 histone tail does not affect the interaction between PHDVC5HCHNSD1 and

C2HRNizp1, whereas in NSD3 binding to H3K9me3 reduces the affinity of PHDVC5HCHNSD3

for C2HRNizp1, via an allosteric mechanism . In conclusion, our data propose a regulative

scenario in which the same NSD protein domain can differently regulate the recruitment of

cofactors necessary for gene transcription.

References:

1. Vougiouklakis T, Hamamoto R, Nakamura Y, Saloura V. Epigenomics. 7(5), 863-74 (2015).

2. Berardi A, Quilici G, Spiliotopoulos D, Corral-Rodriguez MA, Martin-Garcia F, Degano M, Tonon G, Ghitti M,

Musco G. Nucleic Acids Res. 44(7):3448-63 (2016).

3. He C, Li F, Zhang J, Wu J, Shi Y. J Biol Chem. 288(7):4692-703 (2013).

Acknowledgments: Italian Ministry for Health, AIRC, Fondazione Telethon, Fondazione Veronesi.

Presented by: Andrea Berardi

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23

Paramagnetic metalloproteins and fast magic-angle

spinning: theory and experiments meet at the metal

center

Andrea Bertarelloa, Ladislav Benda

a, Kevin Sanders

a, Daniela Lalli

a,

Andrew Pella, Hugh Dannatt

a, Leonardo Gonnelli

b, Roberta

Pierattellib, Isabella Felli

b, Lyndon Emsley

c, Sebastian Wegner

d,

Frank Engelked, Vladimir Pelmentschikov

e, Martin Kaupp

e, Guido

Pintacudaa

aUnivers i té de Lyon, Inst i tu t de Sc iences Analyt iques, (andrea.ber tare l lo@ens - lyon.f r) .

bCERM, Univers ity of Florence, Via L. Sacconi 6, 50019 Sesto F iorent ino.

cEPFL de Lausanne, Inst i tu t des sc iences et ingénier ie chim iques, EPFL SB ISIC LRM,

BCH 1530 (Batochime),CH-1015 Lausanne, Switzer land I taly . dBruker Biospin, Rheinstetten, Germany.

eInst i tu te of Chemist ry , Technische Univers ität Ber l in, Straße des 17. Juni 135, 10623

Ber l in , Germany.

We show how the application of a new set of NMR experiments, recently developed for

the study of complex paramagnetic inorganic battery materials1, can be adapted to the solid-

state NMR analysis of paramagnetic metalloproteins, and can be used to improve the

information obtainable from these systems. These experiments combine ultra-fast (60-111

kHz) magic-angle spinning frequencies and short high-powered adiabatic pulses (SHAPs)2,

and are applied to 13C,15N-labelled microcrystalline, metalloenzyme superoxide dismutase

(SOD), which has two high-affinity binding sites for metal cations3. Here, by the use of the

aforementioned experimental setup and first-principle pNMR calculations4,5, we are able to

detect, characterize and assign 1H, 13C and 15N signals from residues directly coordinating

the metal centers. The present work represents a robust approach to the NMR study of

paramagnetic metalloproteins, opening a new avenue for the study of the structure and the

reactivity of metal centers in complex insoluble systems.

References:

1. Clément, Pintacuda, et al. J. Am. Chem. Soc. 134, 17178-17185 (2012)

2. Kervern G, Pintacuda G., Emsley L. Chem. Phys. Lett. 435, 157-162 (2007)

3. Bertini I, Luchinat C, Piccioli M, Progr in NMR Spectr. 26, 91-139 (1994)

4. Pennanen TO, Vaara J, Phys. Rev. Lett. 100, 133002 (2008);

5. Rouf SA, Mareš J, Vaara J. J. Chem. Theory Comput. 11, 1683–91 (2015)

Presented by: Andrea Bertarello

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24

Structural, dynamical and functional aspects of Ca2+

loaded S100A4 interactions: as seen by NMR

spectroscopy

Gyula Pálfya, Erika Dudás

a, Péter Ecsédi

b, Gergő Gógl

b, László

Nyitrayb, Andrea Bodor

a

aLaboratory of Structura l Chemistry and Bio logy, Eötvös Loránd Universi ty, Pázmány Péter

sétány 1/A, ([email protected] te.hu) . bMotorprote in Structura l Bio logy Laboratory, Eötvös Loránd Univers ity , Pázmány Péter

sétány 1/C, H-1117, Budapest, Hungary .

S100 proteins are vertebrate specific Ca2+ binding proteins of low molecular weight (10-

12kDa) that generally exist as homo- or heterodimers. S100A4 gained increasing attention

because of its metastasis promoting properties and also its important role in the

pathogenesis of rheumatoid arthritis and fibrotic diseases.

Using NMR spectroscopy we extensively investigate the solution characteristics of this

protein in interaction with myosin II isoforms (myo2A and myo2B) and the TAD domain of

p53. A combination of classical and fast acquisition methods is applied for spectral

assignment and secondary structural element determination, backbone dynamics

information is obtained from analysis of relaxation measurements, solvent exchange and

slower exchange contributions are also characterized. Translational diffusion studies help in

following aggregation and determining molecular size.

Our results contribute to the understanding of elevated Ca2+ affinity and high stability of

the S100A4 myosin complex, factors influencing the myosin filament disassembly and

characterization of the disorder-to-order transformation of p53 upon binding.

Acknowledgments: This work was supported by the Hungarian National Research Fund (OTKA) grants

K108437, NK 101072; and the MedInProt program of the HAS.

Presented by: Andrea Bodor

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25

NMR Relaxation and microsecond all-atom Molecular

Dynamics study of the Fluctuations of a Dynamic Protein

Complex

Paloma Gil Rodriguez, ZhenLu Li, Liqun Zhang, Matthias Buck Department of Phys io logy and Biophys ics, Case Western Reserve Univers ity , School of

Medic ine, Cleveland, Ohio 44106, USA. (matth ias [email protected])

It is now recognized that protein-protein interactions in solution are often dynamic,

especially if the binding affinities are only moderately strong. Dynamics originate in part from

the interconversion between structures of the protein complex, e.g. one bound state that is

in equilibrium with one or several alternate configurations. We determined the structure of

such a complex using NMR restraints1 and saw the transitions between different

configurations in microsecond length all-atom molecular dynamics simulations2. Recently,

we also studied the dissociation process of mutant complexes which had a weakened

primary interaction interface. Those simulations suggested that there is no single

dissociation pathway, but that the separation first involves transitions to binding interfaces

with fewer/weaker contacts3. The same molecular dynamics study also revealed that a

substantial change in pico-nanosecond internal protein dynamics accompanies protein

complex dissociation and is a sizeable component of the thermodynamics of dissociation as

an entropy contribution.

Here we have recorded and analyzed relaxation dispersion as well as T1,T2 and hetNOE

relaxation data for 15N nuclei in the mainchain of the EphA2: SHIP2 SAM: SAM complex as

well as in the free proteins. Comparison is made with the microsecond all atom simulations,

also in the context of new simulations of the protein association process. The functional

significance of the protein complex alternate states and their dynamics are discussed.

References:

1. Lee HJ, Hota PK, Chugha P, Miao H, Zhang L, Kim SJ, Alviani RS, Stetzig L, Wang B, Buck M. Structure 20,

41-55 (2012)

2. Zhang L, Buck M. Biophys. J. 105, 2412-2417 (2013)

3. Zhang L, Borthakur S, Buck M. Biophys. J. 110, 877-886 (2016)

Presented by: Matthias Buck

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26

The Distribution of Water and Ions around Nucleic Acids

David A. Case Department of Chemistry and Chemical Bio logy, Rutgers Univers ity , Piscataway, NJ

08854. ([email protected] .edu)

Biomolecules always perturb the solutions into which they are placed. The distribtuions of

water and ions around them can be monintored by a variety of tools, including dialysis,

measurements of partial molar volumes, and X-ray scattering. Computational methods

include explicit solvent simulations and integral equation theories (here, the 3D-RISM

model). I will describe our recent efforts to simulate the distribution of water and ions around

DNA and RNA, including single-strands, duplexes and quadruplexes. An emphasis will be

on the use of such models to interpret X-ray scattering, to small- and wide-angle scattering

in solution, and to scattering from disordered solvent in biomolecular crystals. Estimates of

excess ion and hydration numbers (in solution) and total solvent content (in crystals) can be

compared to experiment as a test of the theory. In turn, a microscopic description of the

molecular environment can yield novel insights into the conformational behavior of nucleic

acids.

References

1. Janowski PA, Cerutti DS, Holton J, Case DA. J. Am. Chem. Soc. 135, 7938-7948 (2013)

2. Giambasu GM, Luchko T, Herschlag D, York DM, Case DA. Biophys. J. 104, 883-894 (2014)

3. Nguyen H, Pabitt SA, Meisburger S, Pollack L, Case DA. J. Chem. Phys. 114, 22D508 (2014)

4. Liu C, Janowski PA, Case DA. Biochim. Biophys. Acta 1850, 1059-1071 (2015)

Presented by: David A. Case

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27

Assembly of Aβ(1-42):Aβ(1-40) mixed fibrils revealed by

SSNMR

Linda Cerofolinia, Enrico Ravera

a, Magdalena Korsak

b, Gianluca

Galloa, Leonardo Gonnelli

a, Marco Fragai

a, Claudio Luchinat

a

a Magnet ic Resonance Center (CERM), Univers ity of Florence, Via L. Sac coni 6, and

Department of Chemistry "Ugo Schif f ", University of F lorence, Via del la Lastrucc ia 3,

50019 Sesto F iorent ino (FI) , I ta ly (cerofo l in [email protected] f i . i t ) . b G iot to Biotech S.R.L. , Via Madonna del Piano 6, 50019 Sesto F iorent ino (FI) , I ta ly .

Formation and propagation of oligomeric aggregates of fragments (such as Aβ(1-42) and

Aβ(1-40)) of the amyloid precursor protein are implied in the onset of Alzheimer’s disease.

Aβ(1-42):Aβ(1-40) ratios higher than the physiological value of 1:9 are critical for

neurotoxicity. Mature fibrils are believed to form as a defense against the permanence of

toxic oligomeric precursors. We have found that fibrils obtained from mixed solutions of

Aβ(1-42) and Aβ(1-40) contain a structurally-uniform 1:1 species, which forms preferentially

from the pure species even when a ratio different from 1:1 is used. Remarkably, this species

differs from both the pure Aβ(1-40) obtained under the same conditions and the recently

reported structures of Aβ(1-42). Formation of stable mixed 1:1 fibrils could represent an

effective way to scavenge the more toxic Aβ(1-42) monomers when their ratio exceeds the

physiological 1:9 value.

Presented by: Linda Cerofolini

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28

Realization of sub-cellular resolution cryogenic ion

microprobe imaging with vitrified samples

Tian Chenga,b

, Florent Planeb, Louise Jensen

a, Stephane Escrig

a,

Bruno M. Humblec, Ben van den Brandt

d, Arnaud Comment

e, Anders

Meiboma,b

a

Ins t i tute of Env ironmental Engineer ing, Ecole Poly technique Federale de Lausanne,

1015, Lausanne, Switzer land. ( t ian.cheng@epf l .ch ) b

Ins t i tute of Earth Sc iences, Univers ity of Lausanne, 1015, Lausanne, Switzer land c

Electron Microscopy Fac i l i ty , Univers ity of Lausanne, 1015, Lausanne, Switzer land d Paul Scherrer Ins t i tu te, CH-5232, Vi l l igen, Switzer land

e Ins t i tute of Phys ics of Bio logical Systems, Ecole Poly technique Federale de Lausanne,

1015, Lausanne, Switzer land

The NanoSIMS 50L instrument is a magnetic-sector, multi-collecting ion probe with ultra-

high spatial resolution (50-100 nm in-plane resolution), increasingly used to study isotopic

and trace element distributions on biological tissue1. Biological samples are normally

prepared for NanoSIMS imaging with standard procedures, involving steps such as

chemical fixation, dehydration, and embedding in epoxy2, which remove most soluble

compounds from the cells. In order to have the capability to study the sub-cellular

distributions of soluble compounds, the conventional NanoSIMS was modified into a

cryogenic variant that we call CryoNanoSIMS, able to maintain vitrified samples around

120K during isotopic imaging. To the best of our knowledge this is the first time a

CryoNanoSIMS has been realized. The experimental protocol and some of the first recorded

CryoNanoSIMS isotopic images will be shown.

References:

1. Hoppe, Cohen and Meibom, GEOSTANDARDS AND GEOANALYTICAL RESEARCH 37(2), 111-154

(2013)

2. Takado Y, Knott G et al, Nanomedicine 11(1), 239 – 245 (2015)

Acknowledgments: We would like to thank Mr. Gilles Grandjean, Mr. Yves Pilloud for their technical help in this

project. This project was supported by the Swiss National Science Foundation (R'Equip 206021_150762)

Presented by: Tian Cheng

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29

An NMR R-factor driving novel Protein Structure

Determination Strategies

Murray Coles Department of Prote in Evolut ion, MPI for Developmental Bio logy, Spemannstrasse 35,

72061, Tübingen, Germany. ([email protected])

For several years we have been incorporating back-calculation of expectation NOESY

spectra into our protein structure determination protocols. Here we put these efforts on a

quantitative footing by introducing a R-factor expressing the ability of a structural ensemble

to predict the experimental data. Our approach is based on the 3D CNH-NOESY1, which

displays contacts to 15N-bound protons in a well-resolved 13C dimension and offers an

excellent compromise between information content and accuracy of back-calculation.

Technical advantages over equivalent spectra with proton dimensions include better

dispersion, more homogeneous linewidths, more complete stereospecific assignments and

lack of uninformative diagonal and water-exchange peaks. RCNH-factors are calculated

directly from the spectrum on a residue-by-residue basis, without user intervention in the

form of peak-picking or cross-peak assignment. We show that they are highly sensitive to

local sidechain and backbone dihedral angles. Moreover, optimization against RCNH can

define the conformers contributing to the structural ensemble and their relative populations.

The use of a quantitative R-factor opens up a range of automated or curated structure

determination strategies where the match between experimental and expectation spectra is

monitored throughout the process. These including fragment assembly approaches that are

ideally suited to integration with data from a wide range of experimental sources.

References:

1. Diercks T, Coles M, Kessler H. J. Biomol. NMR 15, 177-180 (1999)

Presented by: Murray Coles

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30

Protein Interactions in Cells and in Crystals

Peter B. Crowley School of Chemistry , NUI Galway, Univers i ty Road, Galway, IRELAND.

Protein “stickiness” is one of the main

challenges for in-cell NMR. We are interested

in understanding the basis of this stickiness.

GB1 is a “biologically inert” protein that yields

high quality in-cell spectra.1 The introduction of

Arginine residues into GB1 results in a sticky

protein with altered in-cell behaviour.2 A

minimal Arg motif that prevents in-cell detection

will be reported.

We are also investigating the impact of PEGylation on protein structure and assembly.

Recently, we crystallized a model protein that was PEGylated with a single PEG 5000.3 The

protein-polymer conjugate formed a double-helical assembly in the crystal. These helices

were arranged orthogonally to yield a highly porous architecture (see figure). The volume

available in the pores was calculated to be sufficient to accommodate the PEG chains with

random coil conformations. This result suggests that the polymer size can be used to control

protein assembly. Current work on noncovalent PEGylation will also be presented.

References:

1. Crowley PB, Chow E, Papkovskaia T, ChemBioChem 12, 1043-1048 (2011)

2. Kyne C, Ruhle B, Gautier VW, Crowley PB, Protein Sci. 24, 310-318 (2015)

3. Cattani G, Vogeley G, Crowley PB, Nat. Chem. 7, 823-828 (2015)

Acknowledgments: We thank NUI Galway and Science Foundation Ireland for funding.

Presented by: Peter B. Crowley

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31

Dynamics and structural properties of the N-terminal

regions of M. tuberculosis protein kinase G and their role

for the regulation by host induced oxidative stress

conditions

M. Wittwera, Q. Luo

b , V. Kaila

b, and S. A. Dames

a-c

aTechnische Univers i tät München, Depar tment of Chemistry , Biomolecular NMR

Spectroscopy, Lichtenbergstr . 4 , 85747 Garching, Germany bTechnische Univers i tät München, Depar tment of Chemistry , Computat ional Biocata lys is,

L ichtenbergstr . 4 , 85747 Garching, Germany cInst i tu te of Structural Bio logy, Helmholtz Zentrum München, Ingols tädter Landstr . 1,

85764 Neuherberg, Germany. ([email protected])

Mycobacterium tuberculosis escapes killing in human macrophages

by secreting protein kinase G (PknG). The N-terminal 75 residues

were predicted to show no regulatory secondary structure (NORS) and

harbor the only site (T63) phosphorylated in vivo. The following

rubredoxin-like metal-binding motif (RD, 74–147) makes tight

interactions with catalytic domain (148–420) and mediates PknG

redox regulation. C-terminally the kinase domain (KD) is flanked by a

tetratricopeptide repeat domain (TPRD). Here, we present the characterization of the

backbone dynamics and structural properties of the so far uncharacterized NORS and of the

RD in the reduced, metal bound and the oxidized, metal free form by NMR and MD

simulations. Complementary we provide in vitro kinase assay data. The NORS region is as

predicted natively disordered. With respect to the RD, our data suggest that oxidation

induced unfolding of it regulates the substrate access to the catalytic domain and thereby

PknG function under different redox conditions, e.g. if exposed to increased levels of

reactive oxidative species (ROS) in the host macrophages.

Acknowledgments: We thank Dr. N. Scherr and Prof. Dr. J. Pieters from the BIozentrum Basel for some PknG

expression plasmids.

Presented by: Sonja Dames

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32

Targeted labeling of membrane proteins for in-cell solid-

state NMR

Mark Daniëls, Cecilia de Argrela Pinto, Lindsay Baker, Gert Folkers,

Mark Baldus

Department of NMR spectroscopy, Utrecht Univers i ty, Padualaan 8, 3584CH, Utrecht, The

Nether lands. ([email protected])

The study of membrane proteins in their native environment poses additional challenges

for NMR compared to the use of membrane mimetics and synthetic lipids. These difficulties

arise from the need for high levels of protein expression and label incorporation and –at the

same time- the preference to suppress background signals stemming from other molecular

components of the cellular environment1.

Targeted labelling of the protein of interest can serve to alleviate background labelling of

undesired proteins. The antibiotic Rifampicin inhibits the native E.coli RNA polymerase

providing a route to control transcription and subsequent targeted labelling of the gene of

interest under a phage T7 polymerase promoter, which is not inhibited. This approach leads

to vastly reduced background signals in cellular ssNMR spectra as previously demonstrated

for proteins expressed in the inner membrane of bacteria such as KcsA and YidC2. In our

contribution, we show the application of this technique to a multi-protein complex embedded

in the outer membrane, i.e., the BamCDE complex which is critical for functioning of the

BAM insertion machine3.

References:

1. Baldus, M. Biophys J 108, 1585–1586 (2015).

2. Baker, L. A., Daniëls, M., van der Cruijsen, E. A. W., Folkers, G. E., and Baldus, M. (2015). J Biomol NMR

62, 199–208. (2015)

3. Sinnige, T. et al. Structure, 23, 1317-1324 (2015)

Presented by: Mark Daniëls

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33

Mechanism of inhibition and activation of TAp63α in

oocytes

Daniel Coutandin, Christian Osterburg, Jakob Gebel, Sebastian

Kehrloesser, Marcel Tuppi, Katharina Krauskopf, Volker Dötsch a

Ins t i tute of Biophys ical Chemistry , Goethe Univers ity , Max -von-Laue Str 9, 60438

Frankfurt , Germany. ([email protected] - frankfur t .de)

We are investigating the mechanism by which the interaction between two domains that

are unfolded in isolation gain structure and regulate the transcriptional activity of p63. This

protein is expressed in high concentration in oocytes where it serves as a quality control

factor. The C-terminal inhibitory domain interacts with the N-terminal transactivation domain

as well as with the central oligomerization domain and keeps the protein in a dimeric, closed

conformation by forming a β-sheet that blocks the tetramerization interface. The DNA

binding affinity and the transactivation potential is strongly reduced in this inhibited

conformation, explaining why the protein can accumulate to relatively high concentrations

without initiating apoptosis in oocytes. This dimeric and closed conformation is formed co-

translationally and forms a kinetically trapped meta-stable state. We have used a

combination of many methods including NMR and SAXS to build a structural model that

explains the tight regulation of the TAp63α.

References:

1. Deutsch et al, Cell 144:566-76 (2011)

2. Coutandin et al, elife doi: 10.7554/eLife.13909 (2016)

Presented by: Volker Dötsch

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34

Characterization of molecular size and shape by NMR

and SAXS methods

Erika Dudása, Andrea Bodor

a, András Wacha

b, Attila Bóta

b

a Laboratory of Structura l Chemistry and Bio logy, Eötvös Loránd Universi ty, Pázmány

Péter sétány 1/A, 1117 Budapest, Hungary ( [email protected] lte.hu) b Ins t i tute of Mater ia ls and Environmental Chemistry, Research Centre for Natural

Sc iences, Hungar ian Academy of Sc iences , Magyar tudósok körút ja 2. 1117 Budapest,

Hungary .

The diffusion coefficient (D) of a certain molecule under given conditions is closely related

to its size and shape, and it can be measured via PFG-NMR methods. Our previous studies

reported on the diffusion properties of two groups of molecules: folded and disordered

proteins. We provided two equations that allowed estimation of the molecular weight (M),

also the monitoring of aggregation.

After the characterization of the molecular dimension, we investigated the molecular

shape of proteins in the present study. The shape of a molecule can be estimated using the

ratio rG/rH1. This value varies from 0.78 for homogeneous hard spheres to greater than 1.75

for linear polymer chains. The rH (hydrodynamic radii) were calculated from the

experimentally determined diffusion coefficients, while the rG (radius of gyration) values

were determined via SAXS measurements. We examined the effect of various parameters

(protein concentration, temperature, buffer), thus, we were able to find the optimal

conditions to perform NMR and SAXS experiments on the same samples. We studied the

shape and the possible structural changes of lysozyme and calmodulin molecules and

DHPC micelles. The results may bring us closer to the characterization of protein

compactness.

References:

1. Burchard W, Adv. Polym. Sci. 143, 113-194 (1999)

Presented by: Erika Dudás

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35

Characterization of the conformational ensemble of

VirB9CT

in the free state by high-resolution NMR

Denize C. Favaroa, Luciana C. de Oliveira

b, Diorge P. de Souza

b,

Shaker C. Farahb, Roberto K. Salinas

b

aDepar tamento de Química, Ins t i tuto de Ciências Exatas, Universidade Federal de M inas

Gerais , Brazi l . bDepar tment of Biochemistry , Ins t i tute of Chemistry, Univers ity of São Paulo, Brazi l .

([email protected] )

Bacterial secretion systems are composite protein assemblies used for the translocation

of macromolecules across the cell envelope. The Type IV Secretion System (T4SS) is a

versatile macromolecule translocation system used for horizontal transfer of genetic

material, for uptake or release of DNA or, in the case of the phytopathogen Xanthomonas

citri (XAC), for the secretion of toxins that kill other gram-negative bacteria. T4SSs are

generally comprised of a core set of 12 proteins, named VirB1–VirB11 and VirD4. The upper

layer of the conjugative T4SS’s channel consists of fourteen repetitions of an heterotrimer

formed by VirB7 and the C-terminal domains of VirB9 (VirB9CT) and VirB10. Bayliss et al

showed that the pKM101-encoded TraOCT forms a tight and specific complex with the full-

length TraN, respectively VirB9 and VirB7 homologues; and that TraOCT does not suffer

global conformational changes upon binding to TraN1. In contrast, our group showed that

binding of VirB7 from XAC to VirB9CT, involves drastic changes on the conformation and the

dynamics of both partners. In order to obtain insights about the recognition mechanism

between VirB9CT and VirB7, we are studying the conformational ensemble of XAC’s VirB9CT

by NMR. The 1H-15N HSQC spectra of VirB9CT acquired at high temperature (35 ˚C) displays

fewer cross peaks than expected and an heterogeneous distribution of line widths, indicating

that VirB9CT fluctuates among different conformations. A larger number of well-defined cross

peaks becomes visible as the temperature decreases from 35 to 7 ˚C, indicating that a

single conformation predominates at lower temperatures. Analysis of backbone chemical

shifts using TALOS+2 and the Secondary Structure Propensity (SSP)3 algorithm indicated

that the β strands, characteristic of the bound VirB9CT conformation, are present even in the

absence of Xac-VirB7, suggesting that the three-dimensional structure of VirB9CT in the apo

state resembles the bound conformation.

References:

1. Bayliss, R. et al. PNAS. 104, 1673-1678 (2007)

2. Yang S. et al. J. Biomol. NMR. 44, 213-223 (2009)

3. Marsh, J.A. et al. Protein Sci. 15, 2795–2804 (2006)

Presented by: Denize C. Favaro

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36

Integrative Structural Biology of Tetrahymena

Telomerase

Juli Feigon Depar tment of Chemistry and Biochemistry, Univers i ty of Cal i fo rn ia, Los Angeles, PO Box

951569, 90095-1569, Los Angeles, Cal i forn ia, USA. ( fe [email protected] la.edu)

Telomerase is an RNA-protein complex that extends the ends of linear chromosomes,

and is a highly regulated determinant of cellular aging, stem cell renewal, and

tumorigenesis. We are using a combination of NMR spectroscopy, X-ray crystallography,

electron microscopy, mass spectrometry, and biochemistry to study the structure and

function of telomerase. We previously reported the 3D structure of endogenously assembled

Tetrahymena thermophila telomerase holoenzyme at 25 Å resolution using negative stain

electron microscopy. More recently, we determined the cryoelectron microscopy structure of

Tetrahymena telomerase at ~9-Å resolution. In addition to the 7 known holoenzyme

proteins, 2 new proteins are identified, which form a complex (TEB) with single-stranded

telomere DNA-binding protein Teb1, paralogous to heterotrimeric Replication Protein A

(RPA). The p75-p45-p19 subcomplex is identified as another RPA-related complex, CST

(CTC1-STN1-TEN1). This study reveals the paths of TER in the TERT-TER-p65 catalytic

core and ssDNA exit, extensive subunit interactions of TERT essential N-terminal domain,

p50, and TEB, and new subunit identities and structures, including p19 and p45C crystal

structures, providing unprecedented structural and mechanistic insights into telomerase

holoenzyme function.

References:

1. Singh M, Wang Z, Koo BK, Patel A, Cascio D, Collins K, Feigon J. Mol Cell 47, 16-26 (2012)

2. Jiang J, Miracco EJ, Hong K, Eckert B, Chan H, Cash DD, Min B, Zhou ZH, Collins K, Feigon J. Nature 496,

187-192 (2013)

3. Jiang J, Chan H, Cash DD, Miracco EJ, Ogorzalek Loo RR, Upton HE, Cascio D, O’Brien Johnson R,

Collins K, Loo JA, Zhou ZH, Feigon J. Science 350, aab4070 (2015)

4. Feigon J, Chan H, Jiang J. FEBS J. (2016) doi:10.1111/febs.13691 (Epub ahead of print)

Acknowledgments: This work was supported in part by grants from the National Institutes of Health (NIH)

(GM048123) and National Science Foundation (NSF) (MCB1022379) to J.F.

Presented by: Juli Feigon

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37

Macrocyclic paramagnetic agents for magnetic

resonance imaging: an analysis of the determinants of

the observed relaxivity

Alberto Fringuello Mingoa,b

, Sonia Colombo Serraa, Simona Baroni

c,

Claudia Cabellaa, Fabio Tedoldi

a, Silvio Aime

c

aBracco Imaging Spa, Bracco Research Center, Via Ribes 5, 10010, Col leretto Giacosa

(TO), I ta ly , (a lber to.fr [email protected]). b

Dipart imento d i F is ica, Univers i tà degl i Studi d i Tor ino, Via Pietro Giur ia 1, 10100,

Tor ino (TO), I ta ly. c

Dipart imento d i Biotecnologie Molecolar i e Sc ienze del la Salute, Univers ità degl i Studi d i

Tor ino, Via Nizza 52, 10126, Tor ino (TO), I ta ly.

This work aims at pursuing an in-depth relaxometric characterization of two commercial

macrocyclic contrast agents (CAs), Gd-DOTA and Gd-HP-DO3A, by assessing all the

possible contributions acting in physiological conditions (human plasma, 37 °C, pH 7.5).

NMRD profiles, acquired in different media (saline, saline added with albumin, plasma and

ionized simulated body fluid) and fitted according to the inner and outer sphere theoretical

model, lead to the quantification of r1 and of the main factors determining its value. The

increase of r1 in the presence of albumin is similar for the two complexes, but, in plasma, the

two CAs behave quite differently. An increase of about 20-25% (depending on the

frequency) for Gd-DOTA and of 40-50% for Gd-HP-DO3A, respectively, is observed. This

behavior is due to a prototropic exchange contribution for Gd-HP-DO3A (arising at pH 7.5, in

the presence of physiological ionic content) whereas protein binding properties and motion

slowdown due to viscosity are comparable. The prototropic exchange is appreciably efficient

for Gd-HP-DO3A and can be described as an additional inner sphere like process. The

different contributions to relaxivity (protein binding, prototropic exchange and motion slowing

at increased viscosity) were analyzed and treated as additive terms to quantitatively justify

the increase of r1 from saline to human plasma.

References:

1. Laurent S et al. Contrast Med. Mol. Imaging. 1, 128 – 137 (2006)

2. Rohrer M et al. Invest. Radiol. 40, 715 – 724 (2005)

Presented by: Alberto Fringuello Mingo

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38

Structural basis for the interaction between a peptidyl

carrier protein and condensation domain in the

enacyloxin hybrid PKS-NRPS

Angelo Galloa, Daniel Griffiths

a, Paulina K. Sydor

a, Timothy Valentic

b,

Simone Kosola, Shiou-Chuan Tsai

b, Gregory L. Challis

a, Józef R.

Lewandowskia

a Department of Chemistry, Univers i ty of Warwick, Gibbet Hi l l Road, CV4 7AL, Coventry,

UK, (a.gal lo [email protected]) b

Departments of Molecular Bio logy and Biochemist ry, Chemistry, and Pharmaceutica l

Sc iences, Universi ty of Cal i forn ia I rv ine, 517 Bison Ave CA 92697, Irv ine, USA.

Bacteria biosynthesize diverse, structurally-complex products using giant multi-enzymes

known as type I modular polyketide synthases (PKSs) and nonribosomal peptide

synthetases (NRPSs).1 The products of PKSs and NRPSs include antibacterials, anticancer

agents, antifungals, anti-parasitic agents, immunosuppressants and insecticides.

Understanding the molecular mechanisms of PKSs and NRPSs will facilitate their

bioengineering to produce novel drugs and agrochemicals. Here we consider protein-protein

interactions between subunits of a hybrid PKS-NRPS responsible for the biosynthesis of

enacyloxin, an antibiotic that is active against multidrug-resistant Gram-negative bacteria

that cause nosocomial infections.2

Solution NMR studies of apo- and holo-forms of a peptidyl carrier protein (PCP) from the

chain termination module of the enacyloxin PKS-NRPS show that the disordered C-terminus

is responsible for interacting with the condensation domain (CD) that catalyzes chain

release. Consistent with this finding, enzymatic activity is abolished when the C-terminal tail

is removed. Docking calculations based on the solution NMR structure of the PCP and the

X-ray crystal structure of CD provide insights into the interactions between the two proteins.

References:

1. Weissman KJ. Nat Chem Biol. 11(9), 660–70 (2015)

2. Mahenthiralingam E, Song L, Sass A, White J, Wilmot C, Marchbank A, Boaisha O, Paine J, Knight D,

Challis GL. Chem Biol 18(5), 665-77 (2011)

Acknowledgments: ERC-StG-639907

Presented by: Angelo Gallo

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39

Biophysical characterization of PEGylated L-

asparaginase II

Stefano Giuntini, Linda Cerofolini, Enrico Ravera, Marco Fragai,

Claudio Luchinat Magnet ic Resonance Center (CERM), University of F lorence, Via L. Sacconi 6 and

Department of Chemistry "Ugo Schif f ", University of F lorence, Via del la Lastrucc ia 3,

50019 Sesto F iorent ino (FI) , I ta ly.

(g iunt in [email protected] f i . i t )

Bioconjugated biomolecules are extensively employed in medicine and material science

and especially many PEGylated proteins are of medical interest. Since Sixties E. coli L-

asparaginase II (ANSII) has been used as drug for the treatment of acute lymphoblastic

leukemia (ALL)1 and nowadays its PEGylated formulation is routinely commercialized with

the trade name of Oncaspar2. However no satisfactory characterization at atomic detail has

ever been reported. Generally classical techniques cannot be used for PEGylated proteins

because PEGylation prevents crystallization (impeding the use of X-ray analysis) and

furthermore pushes the size beyond the molecular weight limitation for liquid-state NMR.

This work aims at the development of a general method for the biophysical

characterization of PEGylated proteins with a particular interest toward ANSII. After

optimization of the expression, purification and PEGylation conditions, the active tetrameric

structure of PEGylated ANSII has been confirmed by light-scattering analysis and enzimatic

studies. Thus the atomic structure and the preservation of structural integrity after

functionalization has been evaluated by solid-state NMR3.

References:

1. Hill JM, Roberts J, Loeb E, Khan A, MacLellan A, Hill RW. J. Am. Med. Assoc. 202, 882-888 (1967)

2. Graham ML. Adv. Drug Deliv. Rev. 55, 1293-1302 (2003)

3. Ravera E, Ciambellotti S, Cerofolini L, Martelli T, Kozyreva T, Bernacchioni C, Giuntini S, Fragai M, Turano

P, Luchinat C. Angew. Chem. Int. Ed. 55, 1-5 (2016)

Presented by: Stefano Giuntini

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40

Distance distributions between nitroxide spin labels

measured by pulse EPR deliver structural information for

a ~100 kDa protein/RNA complex

Christoph Gmeinera, G. Dorn

b, M. Yulikov

a, F. Allain

b, G. Jeschke

a

a Ins t i tute of Phys ical Chemistry , ETH Zur ich, Vladimir -Prelog-Weg 2, 8093, Zur ich,

Switzer land. (chr is [email protected]) b

Ins t i tute of Molecular Bio logy & Biophys ics, ETH Zur ich, Hönggerbergr ing 64, 8093,

Zur ich, Switzer land.

Site-directed spin labeling (SDSL) in combination with Electron

Paramagnetic Resonance (EPR) pulse dipolar spectroscopy is a

promising tool to obtain long-range structural constraints on

biomacromolecules or large biological complexes1. The alternative

splicing regulator Polypyrimidine Tract Binding Protein 1 (PTBP1),

consisting of four RNA Recognition Motif (RRM) domains, has the

capability to bind to different stem-loops of an Internal Ribosomal

Entry Site (IRES) of an Encephalomyocarditis Virus (EMCV)2. Our

project represents the combination of Nuclear Magnetic Resonance (NMR) and EPR

spectroscopies to determine the structure of this nearly 100 kDa protein/RNA complex.

Here we report on an SDSL approach with nitroxide spin-labeling of the protein and the

RNA. Continuous-wave EPR (CW-EPR) at 9.5 GHz demonstrates high nitroxide labeling

efficiency at several selected sites. Double Electron-Electron Resonance is then used at Q

band (35 GHz) to determine distance distributions between different RRMs in the free state

as well as in the complex with native EMCV IRES, which gives us first data on the

conformation of PTBP1 in this large biomolecular complex.

References:

1. Jeschke G. Annu. Rev. Phys. Chem. 63, 419-446 (2012)

2. Kafasla P, Lin H, Curry S, Jackson R. RNA 17, 1121-1126 (2011)

Presented by: Christoph Gmeiner

r [nm]

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41

In-cell distance measurement by EPR using Gd(III) spin

labels

Daniella Goldfarb Department of Chemical Physics , Weizmann Inst i tute of Sc ience, Rehovot 7610001, Israel.

(Daniel [email protected]. i l )

Spin labeling proteins by site directed spin labeling using Gd(III) chelates holds promise

for in-cell distance measurements by the DEER (double electron-electron resonance)

technique as it can probe variations between in-vitro and in-cell conformations and, in

principle, also follow conformational changes due to interaction with cell components under

various conditions. The advantages of Gd(III) labels stem from their chemical stability in the

reducing environment of the cell, and their high EPR sensitivity at frequencies higher than

Q-band, where one aims at reaching protein concentrations in the range of physiological

concentrations. Accordingly, Gd(III) labels with a small ZFS and a stable and short tether

with a limited flexibility are needed along with the availability of viable cell delivery methods.

To achieve high sensitivity we carry out the measurements at W-band (95 GHz). We used

the small protein ubiquitin as a model to test the applicability of different Gd(III) tags and

their protein conjugation schemes in terms of chemical stability within HeLa cells and

compared the efficiency of cell delivery by electroporation and osmotic shock methods. In

addition, we explored the potential of DEER distance measurements to detect

conformational states of proteins in the cell using Gd(III) labeled calmodulin (CaM). DEER

measurements on several CaM mutants with different Gd(III) tags were first carried out in

vitro on frozen solutions of apo-CaM, Ca2+ bound CaM and Ca2+ bound CaM with a bound

peptide and the expected closed, opened and collapsed conformational states were

distinguished. We then proceeded to carry the measurements in cell extracts and finally in

HeLa cells, where the Gd(III) labeled CaM was delivered into the cells using electroporation

as apo-CaM and Ca2+ bound CaM. The distance distributions obtained in the three different

environment where compared and some differences were observed and will be discussed.

Presented by: Daniella Goldfarb

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42

Fluorine NMR – ligand binding, PREs and other

applications

Angela M. Gronenborn Department of Structura l Biology and Pit tsburgh Center for HIV - Prote in Interact ion,

Univers ity of Pi t tsburgh School of Medic ine, Pit tsburgh, PA, 1526. (amg100@pit t .edu)

Fluorine NMR paramagnetic relaxation enhancement was evaluated as a versatile

approach for extracting distance information in selectively F-labeled proteins. Proof of

concept and initial applications are presented for the HIV-inactivating lectin Cyanovirin-N.

Single F atoms were introduced at the 4-, 5-, 6- or 7 positions of Trp49 and the 4 position of

Phe4, Phe54 and Phe80. The paramagnetic MTSL label was attached to Cys residues that

were placed into the protein at positions 50 or 52. 19F-T2 NMR spectra with different

relaxation delays were recorded and the transverse 19F-PRE rate, 19F-2, was used to

determine the average distance between the F nucleus and the paramagnetic center. Our

data show that experimental 19F PRE based distances correspond to ~0.93 of the 1HN-PRE

distances, in perfect agreement with the gyromagnetic 19F/1H ratio, thereby demonstrating

that 19F PREs are excellent alternative parameters for quantitative distance measurements

in selectively F-labeled proteins.

Presented by: Angela M. Gronenborn

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43

Structure and conformational flexibility of DNA

molecules determined by pulsed EPR spectroscopy

Claudia M. Grytza, Andriy Marko

a, Pavol Cekan

b, Snorri T.

Sigurdssonb, Thomas F. Prisner

a

a Ins t. o f Phys. and Theo. Chem. and BMRZ, Goethe Univers ity , Max-von-Laue-Str . 7,

60438, Frankfur t . Germany (gry tz@pr isner.de)

b

Science Inst . , University of Ice land, Dunhaga 3, 107 Reyk jav ik, Ice land.

Distance measurements between a pair of spin labels attached to

nucleic acids using Pulsed Electron-Electron Double Resonance

(PELDOR also called DEER) is a concomitant tool for structural

biology. If the rigid Ç spin label1 is incorporated pairwise into DNA or

RNA molecules, not only the distance but also the mutual

orientation of the two Ç spin labels can be determined by

multifrequency 2D PELDOR data (X-, Q- and G-band

frequencies)2,3. Thus, the information about the orientation of secondary structure elements

of the nucleic acids can be revealed and used as restraints for structure determination. In

combination with additional knowledge about the topology of the nucleic acid and/or

restraints from other techniques, like NMR, we characterized the conformational space of

flexible DNA motifs, i.e. a bent DNA and a three-way junction, cocaine aptamer4.

References:

1. Cekan P, Smith AL, Barhate N, Robinson BH, Sigurdsson ST. Nucleic Acids Res. 36, 5946-5954 (2008)

2. Schiemann O, Cekan P, Margraf D, Prisner TF, Sigurdsson ST. Angew. Chem. Int. Ed., 48, 3292-3295

(2009)

3. Marko A, Denysenkov V, Margraf D, Cekan P, Schiemann O, Sigurdsson ST, Prisner TF. JACS 133, 13375-

13379 (2011)

4. Grytz CM, Marko A, Cekan P, Sigurdsson ST, Prisner TF. PCCP 18, 2993-3002 (2016)

Presented by: Claudia M. Grytz

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44

Characterization of Mps1 kinase by NMR spectroscopy

Yoshitaka Hirumaa, Marcellus Ubbink

b, Geert J. P. L. Kops

c,

Anastassis Perrakisa

a Div is ion of Biochemistry, Nether lands Cancer Ins t i tute, 1066CX, Amsterdam, The

Nether lands b Leiden Inst i tu te of Chemist ry, Leiden Univers ity , 2300RA Leiden. The Nether lands.

C

Hubrecht Inst i tu te, Royal Nether lands Academy of Arts and Sc iences, 3584CT Utrecht,

The Nether lands. (y.h [email protected] l)

During cell division, all chromosomes have to line up and get attached through their

kinetochores to spindle microtubules. Errors in this process lead to developmental defects

and cancer. The spindle assembly checkpoint (SAC) is a signalling mechanism that inhibits

the progress of cell division until all chromosomes are bipolarly attached to spindle

microtubules. Accumulating evidence shows that the predominant SAC kinase, Mps1 role

directly “senses” by competing with spindle microtubules, whether chromosomes are

attached or not.1,2 My research aims to understand how Mps1 regulates mitosis and the

SAC machinery at the atomic level. Therefore, we have assigned the backbone of the N-

terminal localization module of Mps1. The comparison of the HSQC spectra of the NTE-TPR

(#1−239) and TPR (#62−239) showed significant chemical shift changes in the TPR domain,

indicating the interaction between NTE and TPR.

References:

1. Hiruma Y, Sacristan C, Pachis ST, Adamopoulos A, Kuijt T, Ubbink M, von Castelmur E, Perrakis A, Kops

GJ, Science 348, 1264–7 (2015)

2. Ji Z, Gao H, Yu H, Science 348, 1260–4 (2015)

Acknowledgments: The work is supported by the NWO-VENI grant 5883

Presented by: Yoshitaka Hiruma

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45

NMR study of Coxsackievirus protein 3A

Mark Daniëlsa, Hilde M. van der Schaar

b, Frank J.M. van Kuppeveld

b,

Marc Baldusa, Klaartje Houben

a

a NMR spectroscopy, Bi jvoet Center for Biomolecular Research, Utrecht Univers ity ,

Padualaan 8, 3584 CH Utrecht , The Nether land. (k [email protected])

b

Viro logy, Department of Infect ious Diseases and Immunology, Faculty of Veter inary

Medic ine, Utrecht Univers ity , Yale laan 1, 3584 CL Utrecht, The Nether lands.

Enteroviruses are plus-strand RNA viruses that represent several human pathogens,

such as Polio. The RNA of these viruses codes for one polypeptide chain, which is further

processed into four capsid proteins (VP1-4) and seven non-structural proteins (NSPs). After

virus infection, a virus-induced tubulovesicular network is induced, which forms a platform

for the viral replication complex − a ribonucleoprotein complex containing both viral and

host-cell proteins. The NSP 3A is a critical component of this replication organelle, as it

plays an role in the recruitment of the PI(4)P kinase PI4KB and the Oxysterol binding protein

(OSBP). 3A contains an N-terminal soluble domain, which structure has previously been

determined by NMR for Polio1, and a hydrophobic C-terminus that localizes the protein to

the membrane surface. Mutations in the linker between these two domains as well as in the

hydrophobic domain itself have shown to provide resistance to antivirals that inhibit PI4KB

and OSBP. With this study we aim to determine the topology of 3A in membranes and the

influence of the resistance inducing mutations on the topology and structure of 3A using

both solution and solid-state NMR. For that we have expressed and purified Coxsackievirus

B 3A (CVB3A) and reconstituted in detergents, synthetic lipids and Golgi-like membranes.

Preliminary results suggest that while in LDAO detergent differences in dynamics exist

between the N- and C-terminal domains, most of the protein seems to be membrane

associated in the presence of lipids. Golgi-like lipids further reduce protein dynamics.

References

1. Strauss DM, Glustrom LW, Wuttke DS. J. Mol. Biol. 330, 225-34 (2003)

Presented by: Klaartje Houben

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46

NMR based Structural and Mechanistic investigations of

Histone like DNA binding protein of Helicobacter pylori

Nancy Jaiswala, Nisha Raikwal

a, Krishna Mohan Poluri

b, Dinesh

Kumara

a Centre of Biomedical Research (CBMR), SGPGIMS Campus, R B Road, Lucknow, U.P. -

226014, India. (nancycbmr@gmai l .com) b

Indian Inst i tu te of Technology, Har idwar

Highway, Roorkee, Ut tarakhand- 247667, India.

Histone like DNA binding protein of Helicobacter pylori is a

potential therapeutic target for limiting its infection in humans1. The

pathogen colonizes under harsh acidic and oxidative stress

conditions of human gastrointestinal-tract. This happens because of

a harbinger protein that bacterium expresses to facilitate its

persistent colonization under such harsh conditions, Histone like

DNA binding protein2 (Hup). The in vivo activity of this protein can be

suppressed by the application of small molecule inhibitors.

Working in this framework, the structural and mechanistic investigations on Hup have

been carried out using solution NMR in combination with various biophysical and

computational biology methods. The resulted mechanistic and structural information has

been used further to design small molecule inhibitors following the Structure-based drug

discovery approach. References:

1. Delaney B, Moayyedi P, Forman D. Clin. Evid. 535-548 (2003)

2. Wang G, Lo LF, Maier RJ. DNA Repair (Amst) 11, 733-740 (2012)

Acknowledgments: I would like to thank my supervisor Dr. Dinesh Kumar for guiding me during the course of

this work and our collaborator Dr. Krishna Mohan Poluri for his kind support and suggestions. I would also like

to thank my labmate Nisha Raikwal for helping me.

Presented by: Nancy Jaiswal

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47

Zero-Field Splitting in Gd(III) complexes

Shehryar Khana, Jozef Kowalewski

b, Michael Odelius

a

a Department of Phys ics, Stockholm Univers ity , Albanova Univers ity Center, S -106 91

Stockholm, Sweden. (sherkhan@fys ik .su.se) b

Department of Mater ia ls and Env ironmental Chemist ry, Arrhenius Laboratory, Stockholm

Univers ity , S-106 91 Stockholm, Sweden

The prime objective of contrast agents in Magnetic Resonance Imaging(MRI) is to

accelerate the relaxation rate of the solvent water protons in the surrounding tissue.

Paramagnetic relaxation originates from dipole-dipole interactions between the nuclear

spins and the fluctuating magnetic field induced by unpaired electrons. Currently,

Gadolinium(III) chelates are the most widely used contrast agents in MRI, and therefore it is

incumbent to extend the fundamental theoretical understanding of parameters that drive the

relaxation mechanism in these complexes. Traditionally, the Solomon-Bloembergen-Morgan

equations have been utilized to describe relaxation times in terms, primarily of the Zeeman

interaction, which is the splitting of degenerate energy levels due to an applied magnetic

field. However, in compounds such as Gadolinium(III) complexes with total electron spins

higher than 1 (in this case S=7/2) other interactions such as the Zero-Field Splitting(ZFS)

play a significant role. ZFS is the splitting of degenerate energy levels in the absence of an

external magnetic field. For this purpose, the current research delves into an understanding

of the relaxation process, focusing on ZFS in various complexes of interest, using quantum

chemical methods1.

References:

1. Khan S, Kubica A, Kruk D, Kowalewski J and Odelius M, J. Chem. Phys 142, 034304 (2015)

Presented by: Shehryar Khan

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48

Exploring RNA polymerase regulation by NMR

spectroscopy

Stefan H. Knauer, Martin Strauß, Johanna Drögemüller, Kristian

Schweimer, and Paul Rösch Lehrs tuhl Biopolymere and Forschungszentrum für Bio -Makromoleküle, Univers ität

Bayreuth, Univers itätsstraße 30, 95447 Bayreuth, Germany. (stefan.knauer@uni -

bayreuth.de)

RNA synthesis is a central process in all organisms, with RNA

polymerase (RNAP) as the key enzyme. All cellular genomes are

transcribed by multisubunit RNAPs that are tightly regulated by

numerous transcription factors. Although RNAP of Escherichia coli

(E. coli) has been studied extensively, only little information is

available about its dynamics and transient interactions, which,

however, are crucial for a comprehensive understanding of

transcription regulation in atomic detail.

Here, we present first approaches how E. coli RNAP (~400 kDa) can be studied by

nuclear magnetic resonance (NMR) spectroscopy to gain insights into its dynamic behavior

and its interaction with transcription factors. We developed a highly efficient procedure for

the assembly of active RNAP from separately expressed subunits, allowing specific labeling

of the individual constituents1. We recorded [1H,13C] correlation spectra of Ile, Leu, and Val

methyl groups of complete RNAP and the separately labeled β’ subunit within reconstituted

RNAP, setting the basis for the study of structural changes that might occur upon

transcription factor binding, even in multiprotein complexes1. We further produced all RNAP

subunits individually and established NMR experiments to determine which RNAP subunit a

regulators binds to2. Next, we determined the RNAP binding surfaces of several

transcription factors and followed conformational changes within those factors upon RNAP

binding by NMR spectroscopy using [1H,13C]-labeled methyl groups of Val, Leu, and Ile

within deuterated Nus factors as probes2.

References:

1. Drögemüller J, Strauß M, Schweimer K, Knauer S.H, and Rösch P, Scientific Reports 5,10825-10835 (2015)

2. Drögemüller J, Strauß M, Schweimer K, Jurk M, Rösch P, and Knauer SH, Scientific Reports 5,16428-

16440 (2015)

Presented by: Stefan H. Knauer

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49

Microsecond protein dynamics probed by solid-state

NMR using 15

N R1ρ relaxation experiments under variable

fast MAS conditions (60-110 kHz)

Nils-Alexander Lakomek, Suanne Penzel, Alons Lends, Riccardo

Cadalbert, Matthias Ernst, Beat H. Meier Physical Chemist ry, ETH Zur ich, Vladimir -Prelog Weg 2 ,Zur ich, Switzer land.

(n i ls -a lexander. [email protected])

15N R1ρ relaxation experiments in solid-state NMR are sensitive to time scales and

amplitudes of internal protein motions in the ns-µs time window and can provide valuable

information complementary to solution NMR relaxation dispersion measurements which are

limited to protein dynamics slower than about 10µs and of isotropic nature. Here, using

solid-state NMR 15N R1ρ relaxation experiments with variable MAS frequency under fast

MAS conditions (60-110 kHz), we cover a frequency range of spectral densities which is

wider and more sensitive to ns-µs isotropic and anisotropic motions than in any previous

study. Using a simplified and therefore more robust model-free analysis, we find for the

protein ubiquitin uniform small amplitude dynamics on a time scale of about 1µs across the

entire protein, and larger amplitude motions on the same time scale for several sites,

including the first loop region connecting β-strand 1 and 2, as well as Glu 24, Asn 25 and

Asp 52, which are known to be involved in a hydrogen-bond reordering process, however on

a two to three orders of magnitude slower time scale. Our study adds to the still intense

debate about the presence and/or amplitude of protein backbone dynamics in the ns-µs time

range, recently stirred by Residual Dipolar Coupling (RDC) based experiments and more

recent molecular dynamics simulations, which would directly impact our understanding

about the protein energy landscape and molecular recognition processes. Our data

experimentally establish, with residue-specific resolution, the 1µs time scale for protein

backbone dynamics in the protein ubiquitin. Amplitudes of motion in microcrystalline

ubiquitin (for a time window <100 µs) appear however reduced compared to previous RDC-

based studies in solution NMR (which are sensitive for a time window <10 ms).

Presented by: Nils-Alexander Lakomek

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50

Investigation of the amyloidogenic properties of D76N

beta-2 microglobulin by Solid-State NMR

Tanguy Le Marchanda, Loren Andreas

a, Emeline Barbet-Massin

a,

Hugh H. W. Dannatta,

Michael J. Knight

a, Stefano Ricagno

b, Martino

Bolognesib, Sofia Giorgetti

c, Vittorio Bellotti

c-d, Lyndon Emsley

a and

Guido Pintacudaa

a CRMN, Inst i tu t des Sciences Analyt iques, Univers ité de Lyon, Vi l leurbanne, France.

( tanguy. le.marchand@ens- lyon. fr) b Department of Biotechnology, Univers ity of Mi lano, I ta ly

c Department of Molecular Medic ine, Univers ity of Pav ia, I ta ly

d Centre for Amylo idos is and Acute Phase Prote ins, Univers i ty Col lege London, UK

β-2-microglobulin (β2m), is a 99-residue protein responsible for dialysis-related

amyloidosis, and is recognized as a molecular archetype for the study of folding and amyloid

transition processes. Fibrils of a newly described mutant of β2m (D76N), have been

discovered in kindred patients suffering from gastrointestinal syndromes and autonomic

neuropathy. The aptitude of this mutant to form fibrils in vitro has been revealed to be

spectacularly higher than for the wild type protein. Understanding how a single point

mutation can induce such a change in reactivity would provide a better understanding on the

mechanism of formation of fibrils. Using a combination of ultra-fast (>60 kHz) spinning rates

with 100% NH re-protonation in a perdeuterated background and high magnetic fields, we

acquire spectacularly resolved 1H-detected correlations allowing site-specific investigation of

the backbone dynamics both native and D76N β2m in microcrystalline form. From 1H-15N

dipolar couplings as well as 15N R1 and R1ρ relaxation, we were able to describe nano- to

milli-second dynamics in the two molecules. The comparison of the behaviour of the two

proteins highlights regions of the protein where the D76N mutant is particularly destabilized,

shedding light on a mechanism of formation of the fibrils.

Presented by: Tanguy Le Marchand

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51

Dynamic Nuclear Polarization with High-Fields, Fast-MAS

and High-Temperatures

Moreno Lellia, Sachin R. Chaudhari

b, Pierrick Berruyer

b, David

Gajanb, Anne Lesage

b, Lyndon Emsley

c

a Univers ity of F lorence, Depar tment of Chemistry , Magnet ic Resonance Center (CERM),

Via Luig i Sacconi 6, 50019 Sesto F iorent ino (Florence) , I ta ly. (moreno.le l l i@unif i . i t )

bInst i tu t des Sc iences Analy t iques, Centre de RMN à Très Hauts Champs, Universi té de

Lyon (CNRS/ENS Lyon/UCB Lyon 1), 69100 Vi l leurbanne, France. cInst i tu t des Sc iences et Ingénier ie Chimiques, Ecole Poly technique Fédérale de

Lausanne, 1015 Lausanne, Switzer land.

DNP is a powerful technique to boost sensitivity of MAS NMR

by around 2 orders of magnitude. Experiments are usually

performed at temperatures of ~100 K or lower, with MAS

frequencies not exceeding 15 kHz.1-5 Here we show how the

choice of the solvent and polarizing agent makes it possible to

obtain enhancements of up to 20 @ 273 K and 9.4 T.5 This

approach opens the way to the development of DNP over a wide range of temperatures,

allowing the characterization of dynamics in materials or pharmaceuticals. We also present

some recent results obtained with a fast-MAS DNP probe at high-field (18.8 T, 800 MHz),

and able to spin up to 40 kHz.6

References:

1. Ni QZ, Daviso E, Can TV, Markhasin E, Jawla SK, Swager TM, Temkin RJ, Herzfeld J, Griffin RG. Acc.

Chem. Res. 46, 1933–1941 (2013)

2. Lesage A, Lelli M et al. J. Am. Chem. Soc. 132, 15459−15461 (2010)

3. Lelli M, et al. J. Am. Chem. Soc. 133, 2104−2107 (2011)

4. Rossini A, Zagdoun A, Lelli M, Lesage A, Copéret C, Emsley L. Acc. Chem. Res. 46, 1942 (2013)

5. Lelli M et al. J. Am. Chem. Soc. 137, 14558-14561 (2015)

6. Chaudhari SR et al. PCCP 2016, DOI: 10.1039/c6cp00839a

Acknowledgments: ERC Advanced Grant No. 320860, EQUIPEX contract ANR-10- EQPX-47-01. Frank

Engelke, Christian Reiter and Bruker Biospin are kindly acknowledged for their efforts in developing the 1.3

mm DNP probe.

Presented by: Moreno Lelli

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52

Recent Advances in DNP Enhanced Solid-State NMR at

High Field and Fast MAS

Anne Lesage Univers ité de Lyon, Inst i tut des Sc iences Analyt iques, CNRS/ENS Lyon/UCBL

Over the last few decades, solid-state NMR has developed into an essential analytical

tool to investigate the structure and dynamics of chemical and biological systems. While it

can provide in many cases unprecedented insights into atomic-scale structures, solid-state

NMR suffers from low sensitivity, which strongly limits its application fields. One possibility to

increase the sensitivity of solid-state MAS NMR experiments is Dynamic Nuclear

Polarization (DNP). DNP rely on a transfer of the large polarization of unpaired electrons to

surrounding nuclei upon microwave irradiation. This technique, originally developed for low

magnetic fields, has been shown to be applicable at high magnetic fields, opening new

avenues in the field of materials and structural biology to study molecular systems that were

previously inaccessible to solid)state NMR studies. For complex biological systems, an

order of magnitude increase in sensitivity is now routinely obtained, at 400 MHz and 100 K,

and the technique has been successfully applied on fibrils, membrane-embedded proteins,

virus capsids or whole cell assemblies. In this presentation we will present some recent

experimental developments. In particular we will discuss results from high field (800 MHz)

and fast MAS (~ 40 kHz) DNP NMR. The role of new, highly efficient polarization sources

will be discussed, and we will report some recent high-resolution data obtained on viral

capsid assemblies.

References:

1. Lelli M et al. J. Am. Chem. Soc. 137 (46), 14558–14561 (2015)

2. Chaudhari S et al. Phys Chem Chem Phys. 18, 10616-10622 (2016)

Presented by: Anne Lesage

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53

Methyl groups assignment using paramagnetic effects

with PARAssign

Mathilde Lescannea, Simon Skinner

a, Anneloes Blok

a, Monika

Timmera, Linda Cerofolini

b, Marco Fragai

b, Claudio Luchinat

b,

Marcellus Ubbinka

a Department of Macromolecular b iochemist ry, Univers ity of Leiden, Leiden ins t i tute of

Chemistry , Gor laeus Laborator ies Einste inweg 55, 2333 CC Leiden, The Nether lands.

(m. [email protected] idenuniv .n l) b

Magnet ic Resonance Center (CERM), Univers ity of Florence, Via L. Sacconi 6 , 50019

Sesto Fiorent ino (FI) , I ta ly

The liquid state NMR spectrum assignment of a protein is the first step of any further

study of the dynamics or interactions. It remains challenging for big proteins, where selective

labelling of methyl groups1 is required to obtain well resolved and

sparse spectra. Classical NMR assignment approaches cannot be

used for such an assignment. Using pseudocontact shifts (pcs)

induced by several paramagnetic probes and the X-ray structure of

the protein, PARAssign2 software assigns protein spectrum

requiring datasets from simple 2D-HSQC NMR experiments. The

ability of PARAssign to assign methyl groups has been proved for

simulated pcs. We demonstrate here the ability of PARAssign to

assign with experimental pcs datasets the NMR methyl groups

spectrum of the N-terminal domain of Hsp90 (25 kDa, 78 methyl

groups). The pcs were generated by CLaNP-Yb3+ attached on double cysteine3 on the

surface of the protein via disulfide bridges. Firstly diamagnetic and paramagnetic peaks

were matched to measure the pcs. Then the strategy consisted in running a first time

PARAssign with 23 non ambiguous pcs to get first assignments and a good refinement of

the susceptibility tensors. These tensors were used for further matching of diamagnetic and

paramagnetic peaks to get new pcs used as input for the next run of PARAssign. Finally 55

peaks were assigned and 45 with a high reliability. The completeness of assignments with a

high reliability was consequently 58%.

References:

1. Tugarinov V, Kanelis Y, Kay LE. Nat. Protoc. 1(2), 749-754 (2006)

2. Skinner SP, Mochev M, Hass M, Keizers PHJ, Ubbink M. J. Biomol. NMR 56(4), 401 (2013)

3. Keizers P, Saraglialis A, Hiruma T, Overhand M, Ubbink M. J. Am. Chem. Soc. 130(44), 14802-14812

(2008)

Acknowledgments: The research leading to these results has received funding from the European Union’s

Seventh Framework Programme for research, technological development and demonstration thanks to the

Marie Curie Actions Initial Training Network (ITN) scheme, under grant agreement no 317127, the 'pNMR

project'.

Presented by: Mathilde Lescanne

Susceptibility tensor,

generated by CLaNP-Yb3+

on the N-terminal domain

of hsp90 (pdb:3T0Z)

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54

Toward higher enhancement in Overhauser DNP with

nitroxide radicals

Guoquan Liua, Nikolay Enkin

a, Marcel Levien

a, Niels Karschin

a, Igor

Tkacha, Marina Bennati

a,b

a RG EPR, Max-Planck Inst i tu te for Biophys ical Chemistry , Am Fassberg 11, 37077

Göt t ingen, Germany. (guoquan. l [email protected]) b Department of Chemistry, Univers i ty of Göt t ingen, Tamm anstr . 2, 37077, Gött ingen,

Germany.

We have been investigating the mechanism of Overhauser DNP using nitroxide radicals

as polarizers. In one direction, a series of new nitroxide radicals were chemically

functionalized with a C60 group, which can optically polarize the nitroxide electron spins.

This dynamic electron polarization, quantitatively determined by pulsed EPR, was as high as

about 20 times of Boltzmann polarization. Moreover, these new nitroxide derivatives exhibit

excellent performance in Overhauser DNP at 0.35 T in toluene and higher enhancement

than that using TEMPOL as polarizer was observed under the same conditions. We

examined the saturation factor of these nitroxide derivatives by pulsed ELDOR spectroscopy

and quantitatively interpreted such DNP enhancements. We found that the saturation factor

could increase up to almost unity as a result of enhanced nuclear spin (14N) relaxation in the

nitroxide radicals. High saturation factors and therefore high DNP efficiency can be achieved

at a relatively low polarizer concentration (~ 1.5 mM). These findings shed light on the

design of new polarizers for DNP in liquids.

In another direction, we are investigating Overhauser DNP at high magnetic fields. The

coupling factor, when dipolar interaction dominates the relaxation of target nuclei, usually

decreases dramatically with fields. We are exploring systems where scalar interaction

dominates the relaxation and have observed unprecedented DNP enhancements close to

three orders of magnitudes at 3.35T.

References:

1. Enkin N, Liu G, del Carmen Gimenez-Lopez M, Porfyrakis K, Tkach I, Bennati M. Physical Chemistry

Chemical Physics 17, 11144-11149 (2015)

2. Enkin N, Liu G, Tkach II, Bennati M. Physical Chemistry Chemical Physics 16, 8795-8800 (2014)

Presented by: Guoquan Liu

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55

Enzyme immobilization through Ca/P Biomineralization

Alexandra Loukaa , Enrico Ravera

a , Gil Goobes

b, Marco Fragai

a,

Claudio Luchinata

a Magnet ic Resonance Center (CERM), Univers ity of Florence, Via L. Sacconi 6 and

Department of Chemistry "Ugo Schif f ", University of F lorence, Via del la Lastrucc ia 3,

50019 Sesto F iorent ino (FI) , I ta ly.

( [email protected] f i . i t ) b Department of Chemistry, Univers i ty Bar - I lan, 52900, Ramat Gan, Israel.

HydroxyApatite (HA), is a major component and an essential ingredient of normal bone

and teeth. It is the most stable phase of Ca/P, created by osteoblasts during a well-

orchestrated process, known as Ca/P biomineralization. Several nucleating peptides have

been reported to simulate in situ the biomineralization process1, and have been proposed

for innovative biotechnological applications i.e. thermostable vaccines2 or nano-biomaterials

to be applied in regenerative medicine.

Being inspired by these studies here we have designed an expression vector bearing two

identical Hydroxyapatite Binding Peptides (HABP) with restriction sites in the middle to host

the gene encoding a protein/enzyme of interest. Here we report the in vitro Ca/P

biomineralization of the tetrameric enzyme L-Asparaginase II. The enzyme bearing the

peptide has been expressed and purified and then biomineralized in the presence of

Alkaline Phosphatase and β- Glycero Phosphate. The biomineralized protein is clearly

visible in the solid-state NMR spectra, and the resulting biomaterial retains a sizable

enzymatic activity.

References:

1. Gungormus M, Fong H, Kim IW, Spencer Evans J, Tamerler C, Sarikaya M. Biomacromolecules 9(3), 966-

973 (2008)

2. Wang G, Cao RY, Chen R, Mo L, Han JF, Wang X, Xu X, Jiang T, Deng YQ, Lyu K, Zhu SY, Qin ED, Tang

R, Qin CF. PNAS 110 (19), 7619-7624 (2013)

Presented by: Alexandra Louka

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56

Integrating solution NMR and solid state X-ray structural

data

Claudio Luchinat Magnet ic Resonance Center (CERM), University of F lorence, Via L. Sacconi 6 and

Department of Chemistry "Ugo Schif f ", University of F lorence, Via del la Lastrucc ia 3,

50019 Sesto F iorent ino (FI) , I ta ly ( [email protected] f i . i t )

Long-range NMR restraints, such as diamagnetic residual dipolar couplings and

paramagnetic data, can be used to determine 3D structures of macromolecules. They are

also used to monitor, and potentially improve, the accuracy of a macromolecular structure in

solution by validating or ‘‘correcting” a crystal model. Since crystal structures suffer from

crystal packing forces they may not be accurate models for the macromolecular structures in

solution. However, the presence of real differences should be tested. To achieve this,

simultaneous refinement using X-ray crystallographic and paramagnetic NMR data and/or

diamagnetic residual dipolar couplings has been implemented. Inconsistencies between

crystal structures and solution NMR data, if any, may be due either to structural

rearrangements occurring on passing from the solution to solid state, or to a greater degree

of conformational heterogeneity in solution with respect to the crystal. A decision tree is

proposed1 whereby one can first assess whether the solution and solid state data are

consistent with one another and, if not, assess whether inconsistencies are due to rigid body

displacements of parts of the structure with respect to other parts, as it may be the case for

multidomain proteins. If agreement by rigid body displacement is also not attainable, then

the possibility of mobility in solution is explored by analyzing the conformational space

explored by the molecule.

References:

1. Carlon A, Ravera E, Andrałojć W, Parigi G, Murshudov GN, Luchinat C, Progr. in NMR Spectr. 54, 92-93

(2016) and references therein

Presented by: Claudio Luchinat

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57

Multi-modal interaction between nucleophosmin and

p14ARF monitored by NMR

Enrico Luchinata,b

, Sara Chiarellac, Adele Di Matteo

d, Luca Federici

c,

Maurizio Brunorid, Lucia Banci

a,e

a Magnet ic Resonance Center - CERM, Univers ity of Florence, Via Luig i Sacconi 6, 50019

Sesto Fiorent ino, F lorence, I ta ly. (e [email protected] f i . i t )

b

Department of Biomedical, Exper imental and Cl in ical Sciences “Mario Ser io”, Univers ity

of F lorence, Via le Morgagni 50, 50134, F lorence, I ta ly.

c

Dipar t imento d i Sc ienze Mediche, Oral i e Biotecnologiche, Univers ità d i Chiet i "G.

d'Annunzio", Via dei Vest in i, Chiet i , I ta ly .

d

Dipar t imento d i Sc ienze Biochimiche "A. Ross i Fanel l i " , Sapienza Univers ity of Rome,

00185 Rome, I ta ly . e

Department of Chemistry, Univers i ty of F lorence, Via del la Lastruccia 3, 50019 Sesto

Fiorent ino, F lorence, I ta ly .

Nucleophosmin (NPM1) is an abundant protein mainly localized in the granular

component of the nucleoli. NPM1 is often overexpressed in tumors, and is frequently

mutated in acute myeloid leukemia (AML) where is abnormally localized in the cytosol1. The

N-terminal domain of NPM1 is responsible for its pentamerization and interacts with several

other nucleolar proteins, including the tumor suppressor protein p14ARF. p14ARF is a 14

kDa intrinsically disordered protein involved in cell cycle regulation and in apoptotic

signaling. Here we applied solution NMR to investigate the interaction between (N-ter)NPM1

and p14ARF. Titrating 15N-labeled p14ARF with unlabeled NPM1 reveals a novel interaction

site in addition to the previously known C-terminal binding site2. Multiple sites allow p14ARF

to interact with several copies of (N-ter)NPM1, triggering the formation of high molecular

weight complexes.

References:

1. Di Matteo A, Franceschini M, Chiarella S, Rocchio S, Travaglini-Allocatelli C, Federici L. Oncotarget (2016)

doi: 10.18632/oncotarget.8599 (Epub ahead of print)

2. Mitrea, DM, Grace CR, Buljan M, Yun MK, Pytel NJ, Satumba J, Nourse A, Park CG, Babu MM, White SW,

Kriwacki RW. Proceedings of the National Academy of Sciences 111(12), 4466-4471 (2014)

Presented by: Enrico Luchinat

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58

NMR study of the structure – activity relationship of

Venezuelan equine encephalitis and Hepatitis E viruses’

macro domains

Garyfallia I. Makrynitsaa, Dione Ntonti

a, Konstantinos D. Marousis

a,

Aikaterini C. Tsikaa, Julie Lichière

b, Nicolas Papageorgiou

b, Bruno

Coutardb, Detlef Bentrop

c, Georgios A. Spyroulias

a

a Department of Pharmacy, Univers i ty of Patras, GR -26504, Patras, Greece.

(makryni [email protected])

b

AFMP,UMR7257 CNRS/Aix Marsei l le Univers itè 163 avenue de Luminy, 13288 Marsei l le

CEDEX 09, France. c Ins t i tute of Physio logy I I , Univers i ty of Fre iburg, Fahnenbergplatz, 79085, D-79104,

Fre iburg im Breisgau, Germany.

Macro domains constitute a protein family highly conserved

throughout evolution. Intense research has shown that macro domains

bind ADP-ribose and other NAD+ derived metabolites, suggesting that

they might be involved in several biological pathways1. In the present

study we investigate the conformational and functional properties of two

viral macro domains using molecular biology methods and NMR

spectroscopy2. The 3D NMR solution structure of the macro domain

from Vee virus has been determined with high resolution (Fig. A) while

structural analysis of Hev macro domain is in progress. Furthermore,

NMR driven titration studies were also performed with ADP-ribose for

both macro domains (Fig. B).

References:

1. Han W, Li X. Mutation Research 727, 86-103 (2011)

2. Makrynitsa GI et al. Biomolecular NMR Assignments 9, 247-251 (2015)

Acknowledgments: This work is financially supported by EU FP7-REGPOT-2011 “SEE-DRUG” (nr. 285950)

Presented by: Garyfallia I. Makrynitsa

A

B

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59

Cell-Free Expression of the G Protein-Coupled

Neuropeptide Y2 Receptor

Anika Mantea, Ulrike Krug

a, Rowina Westermeier

b, Frank Bernhard

c,

Peter Schmidta, Daniel Huster

a

a Ins t i tute of Medical Phys ics and Biophys ics , Univers i tät Leipzig, Härte lstraße 16 -18,

04107, Leipzig, Germany.

b Depar tment of Biochemistry, Univers ity of Cambr idge, 80 Tennis Court Road, Cambr idge

CB2, Uni ted Kingdom. c Ins t i tute of Biophys ical Chemistry , Goethe Univers ity , Max -von-Laue Str .9, 60438

Frankfurt /Main, Germany.

The human neuropeptide Y2 receptor (Y2R) is pharmacologically highly relevant, but

detailed knowledge about its structure and dynamics is scarce. Mostly, the major bottleneck

is the inefficient expression due to toxic effects in bacterial expression systems and the low

yield in higher cells. An interesting alternative is cell-free (CF) expression,1 which provides

several advantages for GPCR production in addition to the possibilities to do specific

labeling of amino acids for NMR-studies.2 In this study, the optimization of CF expression of

the Y2R was investigated for targeted NMR studies. We performed the CF expression as

precipitate with an established renaturation protocol.3 The open nature of CF systems allows

a variety of options to manipulate the reaction conditions achieving a high level production.

Y2R could be produced in amounts of 1.64 mg per mL reaction mixture and after

renaturation in DHPC/DMPC bicelles, we could show the functionality of the receptor by

ligand-binding assays resulting which amounted to 100 ± 6%. The main interaction points

between NPY and Y2R are located in an identified binding pocket of the protein.4 In this

area, a Trp is localized likely being involved in ligand binding.4 For this reason, specific 13C-

labeling of all Trp residues was chosen investigating the allocation and function of all Trp in

Y2R by labeling of peptide pairs and point mutation.

References:

1. Spirin et al. Science 242, 1162-1164 (1988)

2. Klammt et al. Eur. J. Biochem. 271, 568-580 (2004).

3. Schmidt et al. Chem. Eur. J. 20, 4986–4992 (2014).

4. Kaiser et al. Angew. Chem. Int. Ed. 54, 7446-7449 (2015)

Presented by: Anika Mante

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60

Dimerization interface and dynamic properties of the

yeast ATPase inhibitor protein IF1 revealed by Site-

Directed Spin Labeling and EPR spectroscopy

Marlène Martinhoa, Nolwenn Le Breton

a, Guillaume Gerbaud

a, Tiona

Andrianaivomananjaonab, Francis Haraux

c, Bruno Guigliarelli

a,

Valérie Bellea

a Aix Marsei l le Univers ité,CNRS,BIP MR 7281, 13402, Marsei l le, France.

(mmart [email protected] .fr )

b

Lifesearch, 72 rue du Fauboug St Honoré, F -75008 Par is, France.

cIns t i tut de Bio logie et de Technologies de Saclay IBITECS, S B2SM, F-91191 Gif sur

Yvet te, France.

Mitochondrial ATPase is regulated by an inhibitory protein called

IF1 which role is to prevent the wasteful hydrolysis of ATP under

anaerobic conditions. The oligomeric state of IF1 related to pH is

crucial for its inhibitory activity. Extensive structural studies have

been performed to characterize bovine IF1 (bIF1) but little is known

concerning the structural organization of yeast IF1 (yIF1). bIF1 is an

inhibitory dimer at low pH and a non-inhibitory tetramer at high pH1.

For yIF1, a monomer/dimer equilibrium exists, high pH values

favoring the monomeric state2. The objective of our study was to

determine the dimerization interface of yIF1 and to gain insights into

the dynamics of its dimeric form using SDSL-EPR, using cw EPR

and pulsed DEER experiments. This work brings the first structural

characterization, at the residue level, of the dimeric form of yIF1 in

solution. This result reveals that the dimeric form of yIF1

corresponds to the non-inhibitory (inactive) state3.

References:

1. Walker JE. Biochem. Soc. Trans. 41, 1-16 (2013)

2. Cabezón E et al. J. Biol. Chem. 277, 41334-41341 (2002)

3. Le Breton N et al. BBA-Bioenergetics 1857, 89-97 (2016)

Presented by: Martinho Marlène

1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0

r(nm)

54

54

IF1 L54CMTSL

IF1 H39CMTSL

3939

(A)

(B)

Figure 1. (A) Inter-spin

distance distribution from Q-

band DEER experiments for

H39CMTSL

and L54CMTSL

at

pH 5.0. (B) Model of yIF1

dimer deduced from the EPR

measurements.

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61

Structural and Dynamical Characterization of gp41 from

HIV by Nuclear Magnetic Resonance

Maya-Martinez R.a,b

, Bougault C. a,b

, Hock M. a,c

, Weissenhorn W. a,c

,

Simorre J-P. a,b

a Ins t i tut de Bio logie Structura le, Grenoble, France.

b Biomolecular NMR Spectroscopy Group, ( rober to.maya-mart [email protected] r ) .

c .Entry and Budding of Enveloped Viruses Group2.

AIDS is considered as one of the first worldwide pandemic caused by the Human

Immunodeficiency Virus (HIV). According to the World Health Organization (WHO report

2015), there were approximately 36.9 million people living with HIV at the end of 2014.

There is no cure for HIV infection, but only some antiretroviral drugs that can control the

proliferation of the virus in the host. In this context, more than one drug is usually

administered at the same time, which targets one of the essential viral proteins like the

reverse transcriptase, the viral protease, the integrase or the surface glycoprotein Env. The

latest protein is of particular interest as it is responsible for the fusion between the viral

particle and the host cell, and as such it became one of the main targets for neutralizing

antibodies and for the design of HIV vaccines. The Env protein (also named gp160) is

constituted of two non-covalently bound subunits gp120 and gp41. During the infection

process gp120 interacts with the host cell receptor and change its conformation, then

releasing gp41 that is essential in the fusion of the host cell and viral membranes. During

the pre-fusion state, gp41 exposes the highly conserved region named membrane proximal

external region (MPER). This region is highly immunogenic and can induce the synthesis of

broad neutralizing antibodies like 2F5, 4E10 and 10E8, which by interacting with MPER

block the transition to fusion state of gp41 with six bundle conformation that bring the two

membranes together. For this reason many studies have focused on designing good

immunogenic gp41 or MPER constructs that would stabilize this particular pre-fusion

conformation and induce the synthesis of neutralizing antibodies for vaccine applications.

None of the structures of chimeric proteins studied so far by X-ray crystallography or

Nuclear Magnetic Resonance (NMR) have reproduced this gp41 pre-fusion state. In this

work, we are trying to characterize by SPR and NMR a gp41int protein constructed which

escaped crystallization so far and is mimicking the intermediate state during the HIV

infection. Our preliminary SPR results show that gp41int is recognized by MPER-specific

antibodies, 10E8 and the new MPER4 antibody. From these results arise our interest to

characterize by NMR the structure and dynamics of this gp41int construct alone and in

complex with the target antibodies. Preliminary NMR data shown that gp41 int is highly

flexible. NMR resonance assignment is in process despite the poor dispersion of the signal

and will allow us to better characterize the dynamics of this pre-fusion intermediate.

Presented by: Roberto Maya-Martinez

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62

Allosteric Coupling in an Ion Channel: Solid State NMR

Studies of KcsA

Ann McDermott Department of Chemistry, Co lumbia Universi ty

Cell signaling in biological systems often involves key proteins in the lipid bilayer

membrane, ion channels being an important example. Messages from the inside of the cell

can be displayed on the outside, and vice versa, due to transmembrane allosteric changes in

conformation and binding affinity. We have focused on allostery in an ion channel, full-length

wild type KcsA channel in a native like membrane environment. New solid-state NMR

methods are applied to obtain atom specific, structural and dynamic information on the

channel opening and, inactivation. An important channel-inactivation process, known as C-

type inactivation, is apparently universal in K+ channels, and partly controls the mean open

time in a number of important channels in the human nervous system, thereby affecting the

fidelity of signals. We recently showed that this activation mechanism has as its central step

the evacuation of ions from the selectivity filter. Although there is still some controversy

regarding this hypothesis, a number of insightful experiments form other laboratories lead to

the same essential model. This slow spontaneous inactivation exhibits the classic signatures

of transmembrane allostery, wherein the activated state is a metastable intermediate of the

allosteric pathway. In ongoing work, we use NMR detected double titration experiments to

show that K+ ion binding on the extracellular selectivity filter is strongly allosterically coupled

to proton binding in the intracellular activation residues, over 30 Å away. These NMR titration

studies also contain important information on the residues involved in the allosteric pathway.

Functional studies of F103 in the hinge of the inner helix suggested an important role for its

bulky sidechain in the inactivation; we recently showed that energetic strength of coupling of

the gates is strongly altered when this residue is mutated to alanine, and characterized other

mutants that are important for inactivation. These results provide quantitative site-specific

measurements of allostery in a bilayer environment, and highlight the power of describing ion

channel gating through the lens of allosteric coupling.

Presented by: Ann McDermott

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63

A hybrid structure approach for the ASC

inflammasome

Beat H. Meiera, Francesco Ravotti

a, Lorenzo Sborghi

b, Adam Mazur

b,

Henning Stahlbergb, Anja Böckmann

c, Sebastian Hiller

b

a Physical Chemist ry, ETH Zur ich, 8093 Zur ich, Switzer land.

b Biozentrum, Univers ity of Basel, 4058 Basel, Switzer land; c Inst i tu te for the Bio logy and

Chemistry of Prote ins, 69367 Lyon, France

We describe the NMR assignment and a

combined Cryo-EM / NMR approach for the

atomic-resolution structure determination of the

mouse inflammasome. “Inflammasomes control

the innate immune response by activating

caspase-1, thus promoting the secretion of

cytokines in response to invading pathogens

and endogenous triggers. The adapter protein

ASC [apoptosis-associated speck-like protein contains a caspase-recruitment domain

(CARD)], consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal

CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1

recruitment.” (from Sborgi et al). We determine the structure of the PYD domain in its fibrillar

form by a hybrid approach with a joint refinement step for EM and NMR data. The CARD

domain is shown, by NMR, to be flexible and well described by a random-coil conformation.

The chances and limitations of the hybrid approach between Cryo-EM and NMR are

discussed.

References:

1. Sborgi L, Ravotti F, Dandey VP, Dick MS, Mazur A, Reckel S, et al. Proceedings of the National Academy of

Sciences of the United States of America 112(43), 201507579–16 (2015)

Presented by: Beat H. Meier

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64

Relationship between structural intrinsic disorder and

enzyme catalysis in UreG: a SDSL-EPR study

Barbara Zambellib, Alessio Bonucci

a, Marta Palombo

b, Stefano

Ciurlib, Bruno Guigliarelli

a, Valérie Belle

a, Elisabetta Mileo

a

a Laboratoire de Bioénergét ique et Ingénier ie des Proté ines, Aix -Marsei l le Univers i té and

CNRS, Marsei l le, France. (emi [email protected] .fr )

b Laboratory of Bio- Inorganic Chemistry , Universi ty of Bologna, Bologna, I ta ly .

Nickel delivery into the active site of urease, an essential enzyme for plants, fungi and

bacteria, requires the presence of four accessory proteins, named UreD, UreF, UreG and

UreE. UreG, responsible for hydrolysis of GTP, exists in solution as an ensemble of

interconverting conformations with significant degree of secondary and tertiary structure. For

these reasons it has been classified as an intrinsically disordered enzyme.1

The aim of this study is to investigate the relationship between of the structural flexibility

of UreG and its activity using Site-Directed Spin Labeling (SDSL) combined with Electron

Paramagnetic Resonance (EPR) spectroscopy. SDSL-EPR has emerged as a powerful

approach to study changes in protein structures and to follow folding/unfolding events that

are not readily amenable with X-ray crystallography. This technique relies on the selective

grafting of paramagnetic labels (e.g. nitroxide radicals) on cysteine followed by the

monitoring of EPR spectra whose shape reflects the mobility of the spin label.2 The

structural behavior and the enzymatic activity of UreG were studied in the presence of

additives that either decrease (i.e. GndHCl, temperature) or increase protein secondary or

tertiary structure (i.e. SDS, TFE, TMAO). CD and NMR were used as complementary

approaches. Taken together, the results demonstrated that the activity of UreG strongly

depends on its structural flexibility: folding-inducers abolish GTP hydrolysis while a

moderately increase of disorder maintains or slightly increases protein catalytic activity.

References:

1. Zambelli B, Cremades N, Neyroz S, Turano, P, Uversky VN, Ciurli S. Mol. Biosyst. 4, 220-228 (2012)

2. Lorenzi M, Sylvi L, Gerbaud G, Mileo E, Halgand F, Walbyrger A, Vezin H, Belle V, Guigliarelli B, Magalon

A. PLOS One 7, e49523, (2012)

Presented by: Elisabetta Mileo

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65

Calcium Impairment and Altered Dynamics of a

Cardiomyopathy-causing Mutation in Troponin C

Explains Disease Phenotype

Adolfo Moraesa, Mayra A. Marques

b, José R. Pinto

c, Anwar Iqbal

b,

Mariana Quezadob, Murilo M. Pedrote

b, Jerson L. Silva

b, Guilherme

A. P. Oliverirab

a Department of Chemistry, Inst i tu te of Exact Sc iences, Federal Univers ity of Minas

Gerais , Av. Antonio Car los ,31270-901, Belo Hor izonte, Brazi l .

(adolfo.dq.ufmg@gmai l.com). b

Ins t i tute of Medical Biochemistry , Nat ional Center of Nuclear M agnet ic Resonance,

Federal Univers i ty of Rio de Janeiro, Rio de Janeiro, Brazi l . c

Depar tment of Medical Sc iences, Col lege of Medic ine, F lor ida State Univers i ty, Rio de

Janeiro, Brazi l .

The most common cardiomyopathies are Hypertrophic (HCM) and Dilated (DCM)

cardiomyopathy, the former being the major cause of sudden death affecting 1 per 500

persons. cTnC is the Ca2+ sensor of sarcomere and plays an important role in regulating

muscle contraction. Although several cardiomyopathy-causing mutations were identified in

cTnC, no information about their structural effects have been attempted to explain HCM

phenotype. Here we showed the mutant D145E inactivates both Ca2+ binding sites at cTnC

C-domain and abolishes the binding to cTnI128-147 peptide because of an altered dynamics

occurring in the µs-ms timescale. To this end, we used an ensemble of thermodynamic and

structural approaches including steady-state fluorescence, circular spectropolarimetry, small

angle X-ray scattering (SAXS) and Nuclear Magnetic Resonance (NMR). Carr-Purcell-

Meiboom-Gill (CPMG)1 relaxation dispersion experiment has captured a low-populated

conformational state as the result of a disease-related mutation that disrupts ion

coordination. Our results may help to explain how altered dynamics may affect protein

functionality and adaptation to the development.

Figure 1: Graphical summary of the proposed mechanism in which the mutation D145E alters properties and

function of cTnC.

References:

1. Korzhnev DM, Kay LE. Acc. Chem. Res. 41, 442-451 (2008)

Acknowledgments: CNPq, CAPES, FAPERJ and IMBEB

Presented by: Adolfo Moraes

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66

Characterization of conformational protein disorder from

NMR chemical shifts

Jakob Toudahl Nielsen, Frans A. A. Mulder Department of Chemistry and iNANO, University of Aar hus, Gustav Wieds Vej 14, 8000,

Aarhus, Denmark. ( [email protected])

There is a great desire to relate the structural properties of intrinsically disordered

proteins (IDPs) and intrinsically disordered regions (IDRs) to biological function and

tendency to aggregate. NMR spectroscopy offers unique capabilities for this: Backbone

chemical shifts are the most sensitive and easily accessible reporters of polypeptide

conformation and dynamics. Differences between measured chemical shifts and reference

values for unstructured (poly)peptides can be utilized to easily assess the tendency for local

structure in IDPs, even if short-lived and only dynamically present. Several years ago we

constructed the first 'random coil' chemical shift database, ncIDP, for this purpose – based

on data for a small subset of 12 proteins1. The ncIDP reference database can be used to

detect and classify areas of order/disorder, and produces residue-specific propensities

towards α-helical or β-sheet-like structure2. Please find it at http://www.protein-nmr.org – as

well as some additional tools useful for the NMR spectroscopist.

I will introduce a new database, CheZOD, with data for proteins with varying degrees of

disorder3. Using a total of 25,369 chemical shifts, we constructed ncIDP2.0, which predicts

the 'random coil' chemical shifts for IDPs better than alternative methods available. I will

present examples of how these tools allow us to accurately investigate the patterns of order

and disorder in proteins. With the experimental NMR data in hand we can finally critically

assess the host of protein disorder predictors developed by bioinformaticians. We find that

these tools demonstrate different levels of success, and we can rank them accordingly.

References:

1. Tamiola K, Acar B, Mulder FAA. J. Am. Chem. Soc. 132, 18000-18003 (2010)

2. Tamiola K, Mulder FAA. Biochemical Society Transactions 40(5), 1014-1020 (2012)

3. Nielsen JT, Mulder FAA. Front. Mol. Biosci. 3(4), 1-12 (2016)

Presented by: Frans A. A. Mulder

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67

Structural basis for PHDVC5HCHNSD1-C2HRNizp1

interaction: implications for Sotos syndrome

Andrea Berardia,b

, Giacomo Quilicia, Dimitrios Spiliotopoulos

a, Maria

Angeles Corral-Rodrigueza,c

, Fernando Martin-Garciaa, Massimo

Deganod, Giovanni Tonon

e, Michela Ghitti

a, Giovanna Musco

a

a Biomolecular NMR Unit , Div is ion of Genet ics and Cel l Bio logy, IRCCS S. Raffaele

Sc ient i f ic Ins t i tute, Mi lan, 20132, I ta ly .

b Univers i tà degl i Studi d i Mi lano, I ta ly .

c Univers i tà Vita e Salute San Raffaele, Mi lano, 21032, I ta ly.

d Biocrysta l lography Uni t , Div is ion of Immunology, Transplantat ion, and Infect ious

Diseases, IRCCS S. Raf faele Sc ient i f ic Inst i tute, Mi lan, 20132, I ta ly.

e Funct ional genomics of cancer , Div is ion of Exper imental Oncology, IRCCS S. Raffaele

Sc ient i f ic Ins t i tute, Mi lan, 20132, I ta ly .

Sotos syndrome is an overgrowth syndrome caused by mutations within the functional

domains of NSD1 gene coding for NSD1, a multidomain protein regulating chromatin

structure and gene expression. PHDVC5HCHNSD1 tandem domain, composed by a classical

(PHDV) and an atypical (C5HCH) plant homeo-domain (PHD) finger, is target of several

pathological missense-mutations. PHDVC5HCHNSD1 is also crucial for NSD1-dependent

transcriptional regulation and interacts with the C2HR domain of transcriptional repressor

Nizp1 (C2HRNizp1) in vitro. To get molecular insights into the mechanisms dictating the

patho-physiological relevance of the PHD finger tandem domain, I. we solved its solution

structure and provided a structural rationale for the effects of 7 Sotos syndrome point-

mutations; ii characterized its binding to histone H3 peptides and iii. to C2HRNizp1 by ITC and

NMR. We observed only very weak electrostatic interactions with histone H3 N-terminal

tails, conversely we proved specific binding to C2HRNizp1. and generated a 3D model of the

complex, corroborated by site-directed mutagenesis. We suggest a mechanistic scenario

where NSD1 interactions with cofactors such as Nizp1 are impaired by PHDVC5HCHNSD1

pathological mutations, thus impacting on the repression of growth-promoting genes, leading

to overgrowth conditions.1

References:

1. Berardi et al. Nucleic Acids Research 44, 3448-3463 (2016)

Acknowledgments: Fondazione Veronesi, Ministero della Salute RF-2013-02354880 , AIRC-IG17468, Marie

Curie Co-Fund

Presented by: Giovanna Musco

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68

NMR characterization of integrin αVβ6/Chromogranin

A39-63 interaction

Francesca Nardellia,b

, Giacomo Quilicib, Flavio Curnis

c, Martina

Fiocchic, Angelo Corti

c, Giovanna Musco

b

a Univers ità Vita-Salute San Raffaele, Via Olgett ina 58, 20132, Mi lan, I ta ly.

(nardel l i . f rancesca@hsr. i t ) b

Biomolecular NMR Laboratory , IRCCS S an Raf faele, Via Olget t ina 60, 20132, Mi lan,

I ta ly. c

Tumor Bio logy and Vascular Target ing, IRCCS San Raf faele, Via Olgett ina 60, 20132,

Mi lan, I ta ly .

αVβ6 integrin is an important target for both imaging and treatment, since it is up-

regulated in many types of cancer, but not in normal healthy organs1. The crystal structure

of αVβ6 bound to a short peptide of TGF-β3 containing the RGDLXXL motif, defining the

selectivity towards αvβ6, reveals that the RGD motif adopts a loop conformation, whereas

the LXXL sequence adopts an α-helical structure2. Also Chromogranin A selectively binds

to αvβ6; in particular, its shorter peptide CgA39-63, which contains the RGDEXXL motif

similar to the RGDLXXL, binds tightly to αvβ63. We solved the solution structure of CgA39-63

by NMR; we found that the RGD sequence is non-structured, whereas the EXXL sequence,

similarly to TGF-β3 peptide when bound to αvβ6, adopts an α-helix. In order to get structural

insights into peptide/integrin interaction, we have performed 1D-STD experiments between

CgA39-63 and αVβ6 extracellular domain, which confirmed their binding. We are currently

repeating STD experiments using human cancer cells expressing αVβ6, for the

characterization of the interactions in a more physiological context4. We also plan to perform

2D-QHSQC-STD experiments using the fully labeled peptide,5 in order to get a reduced

signal overlap to better define the ligand epitope mapping.

References:

1. Bandyopadhyay A, Raghavan S. Drug Targets 10, 645-652 (2009)

2. Dong X, Hudson NE, Lu C, Springer TA. Nat. Struct. Mol. Biol. 21, 1091-1096 (2014)

3. Curnis F, Gasparri A, Longhi R, Colombo B, D’Alessio S, Pastorino F, Ponzoni M, Corti A. Cell Mol. Life Sci

69, 2791-2803 (2012)

4. Mari S, Invernizzi C, Spitaleri A, Alberici L, Ghitti L, Bordignon C, Traversari C, Rizzardi G, Musco G.

Angew. Chem. Int. Ed. 49, 1071-1074 (2010)

5. Sorge JL, Wagstaff JL, Rowe ML, Williamson RA, Howard MJ. Org. Biomol. Chem. 13, 8001-8007 (2015)

Acknowledgments: AIRC IG17468, Ministero della salute (RF-2011-02350836)

Presented by: Francesca Nardelli

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69

[2Fe-2S] BOLAs-GLRX5: novel players in ISC assembly

machinery

Veronica Nastaa, Marta A. Uzarska

b, Benjamin D. Weiler

b, Farah

Spantgarb, Simone Ciofi-Baffoni

a, Maria R. Saviello

a, Leonardo

Gonnellia, Ulrich Mühlenhoff

b, Lucia Banci

a, Roland Lill

b

a Magnet ic Resonance Center CERM, University of F lorence , Via Luig i Sacconi 6, 50019,

Sesto Fiorent ino, F lorence, I ta ly and Depar tment of Chemistry, Universi ty of F lorence ,

Via del la Lastrucc ia 3, 50019 Sesto F iorent ino, F lorence, I ta ly . ([email protected] f i . i t ) bInst i tu t für Zytobiologie und Zytopathologie, Phi l ipps -Univers ität , Robert -Koch-Str . 6,

35032 Marburg, Germany and LOEWE Zentrum für Synthet ische Mikrobio logie SynMikro,

Hans-Meerwein-Str . , 3504 Marburg, Germany.

Assembly of mitochondrial iron-sulfur (Fe/S) proteins is a key process of cells. In the first

phase of this pathway, formation of a [2Fe-2S] cluster in the human mitochondrial matrix

occurs on a scaffold protein (ISCU). The cluster is then released to monothiol glutaredoxin 5

(GLRX5), which was shown to mediate the transfer of [2Fe-2S] clusters from the scaffold

protein to several target apo proteins. The second phase is dedicated to the assembly of

[4Fe-4S] proteins, yet this part is poorly understood. In this frame BolA-like proteins, which

are grouped into three subfamilies BolA1-, BolA2-, and BolA3-like proteins, have recently

emerged as novel players. In humans, mutations in the mitochondrial BOLA3 cause a

severe disease associated with defects in the Fe/S proteins lipoate synthase and respiratory

chain complexes I and II. These findings suggested that mitochondrial BOLA proteins might

play a crucial role in the maturation of Fe/S proteins.

Here, we determined the 3D structures of hBOLA3 and hBOLA1 proteins and

investigated protein-protein interaction between apo hBOLA3 (or apo hBOLA1) and both

apo and [2Fe-2S] GLRX5 form by NMR. Collectively, the data showed that both proteins

interact with GLRX5 forming a 1:1 hetero-complex in both apo and [2Fe-2S] states. The

functional role of these complexes in Fe/S protein biogenesis is now under investigation.

Presented by: Veronica Nasta

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70

Molecular mechanism of spider silk formation and

structural studies of artificially spun fibers

Martins Otikovsa, Gefei Chen

b,c, Marlene Andersson

d, Kerstin

Nordlingb, Michael Landreh

e, Qing Meng

c, Hans Jörnvall

e, Nina

Kronqvistb, Anna Rising

b,d, Jan Johansson

b,d,f, Kristaps Jaudzems

a

aLatv ian Ins t i tute of Organic Synthes is, Latv ia. (mao@osi. lv)

bDepartment of Neurobio logy, Care Sc iences and Society, KI , Sweden.

cIns t i tute of Bio logical Sc iences and Biotechnology, Donghua Univers ity , China.

dDepar tment of Anatomy, Phys io logy and Biochemistry , SLU, Sweden.

eDepar tment of Medical Biochemistry and Biophys ics, KI , Sweden.

fInst i tu te of Mathematics and Natura l Sc iences, Tal l inn Univers ity , Estonia.

Spider silk is one of the most outstanding biomaterials, combining high tensile strength

with elasticity and toughness. It is made through association of spider silk proteins, called

spidroins, which have a MW of ca. 300-600 kDa and >90% of its sequence is composed by

repetitive domains (Rep). Rep domains confer mechanical properties to spun fibers and are

flanked by N- and C-terminal domains (NT and CT). The spinning process of fibers from

highly concentrated spinning dope (30-50 weight%) is tightly controlled by NT and CT to

ensure rapid fiber production and at the same time prevent premature association of

spidroins in storage sac, where spinning dope is kept or before reaching exit of S-shaped

duct connecting storage sac to outer environment.

Here we present a molecular mechanism, how NT controls polymerization of spidroins.

Determination of solution state NMR structures of monomeric and dimeric NT from spidroins

expressed in A. ventricosus minor ampullate silk gland, reflecting conformation of NT before

and after spidroins have assembled into fibers, respectively. These structures together with

site-directed mutagenesis studies and Trp fluorescence spectroscopy have allowed us to

identify key residues ensuring monomer-dimer transition. Additionally, we characterized in

solution a spidroin construct that consists of NTD, two Rep and CTD and studied structure

of each of the domains by solid state NMR in fibers spun from NT-2Rep-CT construct.

Acknowledgments: Financial support from 7th Framework Programme project INNOVABALT is gratefully

acknowledged.

Presented by: Martins Otikovs

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71

tert-Butyl Groups: Chemical NMR Labels for Large and

Difficult Proteins

Wan-Na Chena, Christoph Nitsche

a,b, Kala Bharath Pilla

a, Luke A.

Adamsc, Shereen Jabar

a, Christian D. Klein

b, Bim Graham

c, Thomas

Hubera, Gottfried Otting

a

a Research School of Chemistry , Austra l ian Nat ional Univers ity , ACT 2605, Canberra,

Austra l ia . (got t fr ied.ot t [email protected]) b

Ins t i tute of Pharmacy and Molecular Biotechnology, Heidelberg Univers i ty, Im

Neuenheimer Feld 364, 69120 Heidelberg, Germany. c

Monash Inst i tu te of Pharmaceut ica l Sciences, Monash Univers ity , VIC 3052, Parkv i l le ,

Austra l ia .

Tert-butyl groups present outstanding chemical probes for NMR

spectroscopy. They can be found in chemically synthesized ligand

molecules and can be introduced into proteins with an unnatural

amino acid. For example, O-tert-butyl-tyrosine (Tby) can be site-

specifically incorporated into proteins using established orthogonal

aminoacyl-tRNA synthetase/tRNA systems. Tert-butyl groups

deliver an exceptionally narrow and tall 1H NMR signal, and also

produce very intense NOESY cross-peaks. The signals are observable in high molecular

weight systems (> 300 kDa) without isotope labelling. NOESY cross-peaks with the tert-

butyl group of a ligand allow probing of the structure of a stable protein-ligand complex

without prior resonance assignments of amino acid side chains. Pseudocontact shifts

generated by paramagnetic lanthanide tags attached to the protein can be used to locate

the tert-butyl group and its orientation on the protein.

References:

1. Chen WN, Kuppan KV, Lee MD, Jaudzems K, Huber T, Otting G. J. Am. Chem. Soc. 137, 4581-4586 (2015)

2. Chen WN, Nitsche C, Pilla KB, Graham B, Huber T, Klein CD, Otting G. J. Am. Chem. Soc. 138, 4539-4546

(2016)

Presented by: Gottfried Otting

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72

How can heavy chain phosphorylation participate at the

regulation of non-muscle myosins function?

Gyula Pálfya, Péter Ecsédi

b, László Nyitray

b, Andrea Bodor

a

aLaboratory of Structura l Chemistry and Bio logy, Eötvös Loránd Universi ty, Pázmány Péter

sétány 1/a. , H-1117, Budapest, Hungary . bMotorprote in Structura l Bio logy Laboratory, Eötvös Loránd Univers ity , Pázmány Péter

sétány 1/c ., H-1117, Budapest, Hungary

Non-muscle myosin II A and B (NMIIA and NMIIB) heavy chains are long, coiled-coil

structure proteins involved in several aspects of cell migration. Phosphorylation by casein

kinase 2 (CK2) of C-terminal disordered tailpiece and binding of metastatic S100A4 protein

probably alters filament formation and function differentially for the two isoforms. Our aim

was to investigate the effect of phosphorylation on the myosin filaments using NMR

spectroscopy. The C-terminal tailpiece fragments of NMIIA, NMIIB and a chimera protein,

NMIIA coiled coil with NMIIB C-terminal tailpiece was studied (M111A, M124B, M124AB,

respectively). Phosphorylation was monitored in situ by NMR methods and compared with

MS. Assignment showed no significant structural differences in native and phosphorylated

fragments, however the number of phosphorylation sites and kinetics differ. Diffusion NMR

studies showed that M111A and M124AB are dimers, while M124B forms mostly tetramers.

We detected slightly different aggregation tendencies in NMIIB phosphorylated fragments,

which is in good agreement with SAXS results. We conclude that while metastatic S100A4

protein affects only NMIIA filaments, phosphorylation by CK2 kinase regulates only NMIIB

filaments. However, the heavy chain phosphorylation-dependent regulation is not occurring

via intramolecular but rather intermolecular changes affecting electrostatic interactions

between neighboring myosins.

References:

1. Dulyaninova NG, Bresnick AR. Bioarchitecture 3(4), 77-85 (2013)

Presented by: Gyula Pálfy

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73

How to improve the accuracy of solution structures

through refinement of solid state structures?

Azzurra Carlon, Enrico Ravera, Giacomo Parigi, Claudio Luchinat

Magnet ic Resonance Center (CERM), University of F lorence, Via L. Sacconi 6 and

Department of Chemistry "Ugo Schif f ", University of F lorence, Via del la Lastrucc ia 3,

50019 Sesto F iorent ino (FI) , I ta ly. (par [email protected] f i . i t )

Obtaining a complete NMR data set for protein structure characterization is very time

consuming and often difficult in the case of high molecular weight systems. If available,

crystallographic structures are thus commonly used as models for the protein structures also

in solution. However, crystal structures can suffer from crystal packing forces, so that they

may not be accurate models in solution. The paramagnetic restraints (pseudocontact shifts

and residual dipolar couplings), as well as the diamagnetic residual dipolar couplings, can

be used for validating and possibly improving their accuracy. To this purpose, the program

REFMAC5 from CCP4 was modified to allow for the simultaneous use of X-ray

crystallographic data and paramagnetic NMR data and/or diamagnetic residual dipolar

couplings1-3. REFMAC-NMR refinements can thus assess whether experimental NMR data

can be explained by X-ray structural models within the accuracy of their diffraction patterns,

and, in these cases, can provide conformations that comply with both types of restraints.

Inconsistencies between crystal structures and solution NMR data, if any, may be due

either to structural rearrangements occurring on passing from the solid state to solution, or

to a larger conformational heterogeneity. In the case of multidomain proteins, the

paramagnetic restraints can provide the correct reciprocal position of the domains in

solution4, as well as information of the conformational variability experienced by the protein5.

References:

1. Rinaldelli M, Ravera E, Calderone V, Parigi G, Murshudov GN, Luchinat C. Acta Cryst. D 70, 958-967

(2014)

2. Carlon A, Ravera E, Andrałojć W, Parigi G, Murshudov GN, Luchinat C. Progr. in NMR Spectr. 92-93, 54-70

(2016)

3. Carlon A, Ravera E, Hennig J, Parigi G, Sattler M, Luchinat C. J. Am. Chem. Soc. 138, 1601-1610 (2016)

4. Bertini I, Kursula P, Luchinat C, Parigi G, Vahokoski J, Willmans M, Yuan J. J. Am. Chem. Soc. 131, 5134-

5144 (2009)

5. Ravera E, Salmon L, Fragai M, Parigi G, Al-Hashimi H, Luchinat C. Acc. Chem. Res. 47, 3118-3126 (2014)

Presented by: Giacomo Parigi

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74

PFG NMR Investigations of Protein Diffusion in Gels

Reveals Intrinsic Anomalous Behavior for BSA but not

for IL-8

Anja Penk, Daniel Huster Ins t i tute for Medical Phys ics and Biophys ics , Leipzig Univers i ty, Härte ls tr . 16/18, 04107,

Leipzig, Germany.

([email protected] - le ipzig.de) .

Proteins are often used to enhance the performance of an implant or a gel in trauma

surgery and wound treatment. Hence, the controlled release and the local concentration in

early stages of the healing process is of major impact and determined by diffusion.

Depending on the purpose, a huge variety of different proteins can be used for treatment,

e.g. growth factors or anti-inflammatory cytokines, which may differ in molecular weight and

isoelectric point.

To elucidate the influence of these properties, we studied the diffusion of IL-8, a protein

dimer with 18 kDa in total, and BSA in different gels (agarose and collagen) by Pulsed Field

Gradient (PFG) NMR. As expected, the apparent diffusion coefficient indicates hindered

diffusion in a gel environment. In more detail, the apparent diffusion coefficient is decreased

by higher gel concentrations and well described by the Ogston-model1,2.

Furthermore, we obtained the diffusion to be anomalous for BSA but not for IL-8.

Interestingly and in contrast to the apparent diffusion coefficient, the strength of this effect

depends only on the protein and neither on the type of the surrounding isotropic gel nor on

the gel concentration. Thus, we conclude that the strength of anomalous diffusion – which is

expressed by a stretching parameter derived from fractional calculus of the Bloch equations

– reflects an intrinsic protein property. Eventually, possible reasons for this behavior as well

as future applications will be discussed.

References:

1. Ogston AG. Trans Faraday Soc. 54, 1754 – 1757 (1958)

2. Ogston AG, Preston BN and Wells JD. Proc Math Phys Eng Sci, 333, 297 – 316 (1973)

Presented by: Anja Penk

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75

SS-NMR characterization of the sweet protein MNEI in its

native and fibrillar state

Andrea Picaa, Kristaps Jaudzems

b, Jan Stanek

b, Loren B. Andreas

b,

Serena Leonea, Guido Pintacuda

b, Delia Picone

a

a Department of Chemical Sc ienc es, Univers i ty of Naples Feder ico I I , Via Cint ia, I -80136,

Naples, I ta ly, (andrea.p ica@unina. i t) . b

Centre de RMN à Très Hauts Champs, Inst i tut des Sc iences Analy t iques, UMR

5280/CNRS, ENS Lyon, UCB Lyon 1, Université de Lyon, Vi l leurbanne, France.

MNEI is a single chain derivative of the sweet protein monellin,

with promising potential as a sweetener for food and beverage

applications. The structure of the protein is well known and has

been characterized by both x-ray crystallography and solution-

state NMR.1,2 Despite its very high stability to temperature and

chemical agents, its high tendency to form insoluble aggregates

hampers the straightforward use as a food additive.3 The

mechanism by which MNEI aggregates is still unclear but it is

reported that under appropriate conditions the aggregates can

assume a fibrillar organization.4 Here we study MNEI in its fibrillar

and, by comparison, native form by solid-state NMR under fast

(60-100 kHz) magic-angle spinning, in order to get insights on the

structure of the aggregate and on their mechanism of formation.

This data would be extremely helpful to intervene by a

mutagenesis-based approach to improve the performance of the

sweet protein.

References:

1. Hobbs JR, Munger SD, Conn GL. Acta Crystallogr F. 62, 162-167 (2007)

2. Spadaccini R, Crescenzi O, Tancredi T, De Casamassimi N, Saviano G, Scognamiglio R, Di Donato A,

Temussi PA, J Mol Biol. 305, 505-514 (2001)

3. Rega MF, Di Monaco R, Leone S, Donnarumma F, Spadaccini R, Cavella S, Picone D. Food Chem. 173,

1179-1186, (2015)

4. Konno T, Murata K, Nagayama N. FEBS Lett. 454, 122-126 (1999)

Acknowledgments: We thank Fondazione con il Sud (PDR-10 2011) for financial support.

Presented by: Andrea Pica

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76

Fully protonated proteins and proton-detected NMR

at > 100 kHz magic-angle spinning

Loren B. Andreasa, Kristaps Jaudzems

b, Jan Stanek

a, Daniela Lalli

a,

Andrea Bertarelloa, Tanguy Le Marchand

a, Diane Cala-De Paepe

a,

Svetlana Kotelovicab, Inara Akopjana

b, Benno Knott

c, Sebastian

Wegnerc, Frank Engelke

c, Anne Lesage

a, Lyndon Emsley

a,d, Kaspars

Tarsb, Torsten Herrmann

a, Guido Pintacuda

a

a Univers ité de Lyon, Vi l leurbanne, France. (guido.p intacuda@ens - lyon.fr)

b Biomedical Research and Study Centre, Riga, Latv ia.

c Bruker Biospin Rhe instetten, Germany.

d EPFL Lausanne, Switzer land.

Protein structure determination by proton detected magic-angle

spinning (MAS) NMR has focused on highly deuterated samples, in

which only a small number of protons are introduced and

observation of signals from side-chains is extremely limited. Here

we show in fully protonated proteins that at 100 kHz MAS and

above, spectral resolution is high enough to detect resolved

correlations from amide and side-chain protons of all residue types,

and to reliably measure a dense network of 1H-1H proximities that

define a protein structure. Additionally, we find that narrower proton

resonance lines, longer coherence lifetimes and improved magnetization transfer offset the

reduced sample size at 100 kHz spinning and above. Less than two weeks of experiment

time and a single 0.5 mg sample is sufficient for the acquisition of all data necessary for

backbone and side-chain resonance assignment and structure determination. These

findings increase the impact of solid-state NMR to samples that cannot easily be deuterated,

and for samples that can only be produced in sub milligram quantity, and we show examples

of a microcrystalline protein, a protein dimer in an intact viral capsid assembly, and a

membrane protein in lipid bilayers. We expect the technique to pave the way for atomic-

resolution structure analysis applicable to a wide range of samples of high biological

relevance.

Presented by: Guido Pintacuda

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77

Probing the supramolecular structure of the 200 kDa β-

barrel assembly machinery complex by solid-state NMR

Cecilia Pinto, Deni Mance, Mark Daniëls, Markus Weingarth, Klaartje

Houben, Marc Baldus NMR Spectroscopy, Bi jvoet Center for Biomolecular Research, Department of Chemistry ,

Facul ty of Sc ience, Utrecht Univers i ty, Padualaan 8, 3584 CH Utrecht , The Nether lands

Protein folding and insertion into cellular membranes, which is essential for physiological,

pathogenic and drug resistance functions1, requires complex molecular protein machines. In

E.coli the precursors of outer-membrane β-barrel proteins (OMP) are synthesized in the

bacterial cytoplasm. The unfolded precursor proteins are recognized and translocated

across the inner membrane by the Sec translocase. Insertion into the outer membrane is

subsequently coordinated by the β-barrel assembly machinery (BAM2) consisting of the core

component BamA and accessory lipoproteins (BamB, BamC, BamD, and BamE). Recently,

structures were made available of the complete 200kDa complex in detergents3. In spite of

the valuable insights provided by these structures, atomic information on how BAM

dynamically assembles in membranes and how substrate insertion takes place is still

elusive.

Previously, we have shown how NMR can be used to study structure and dynamics of the

core component, BamA, in lipid membranes4, 5. Here we describe a solid-state NMR based

approach to probe contacts between BamA, and subcomplex BamCDE in a membrane

environment and we compare our findings to recent X-ray structures3.

References:

1. Bos MP, Robert V, Tommassen J. Annual Review of Microbiology. 61, 191-214 (2007)

2. Voulhoux R et al. Science. 299, 262-265 (2003)

3. Gu Y et al. Nature. 531, 64-69 (2016)

4. Sinnige T et al. Structure. 23, 1317-1324 (2015)

5. Sinnige T et al. Journal of Molecular Biology. 426, 2009-2021 (2014)

Presented by: Cecilia Pinto

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78

Characterization of XIAP-BIR1 in mammalian cells by

NMR

Panagis Polykretisa, Enrico Luchinat

a, Xuncheng Su

b and Lucia

Bancia

a Magnet ic Resenance Center (CERM), Univers ity of Florence, Via Luig i Sacconi 6, 50019,

Sesto Fiorent ino (FI) , I ta ly. (polykret [email protected] f i . i t ) b

Ins t i tute of Elemento-organic Chemistry , Nankai Univers i ty, Wei j in Road 94, T ianj in ,

China.

X-linked Inhibitor of Apoptosis Protein (XIAP) is a multifunctional metalloprotein,

ubiquitously expressed in human cells except peripheral blood leukocytes. While its primary

function is to block programmed cell death by caspase inhibition, XIAP also plays a critical

role in copper homeostasis and in NF-κB activation. XIAP is frequently overexpressed in

tumors, in which it potentiates cell survival and resistance to anticancer drugs. In particular,

its BIR1 domain, after dimerization, interacts with TAK binding protein 1 (TAB1), which in

turn recruits Transforming Growth Factor β activated kinase 1 (TAK1), with consequent

activation of NF-κB and cell survival pathways. Consequently, the impairment of the BIR1-

TAB1 assembly would have a synergistic action to the pro-apoptotic chemotherapeutics.

The aim of this study is to characterize through NMR the metallation and oxidation state of

BIR1 and its D71N/R72E mutant in HEK293T cells, in order to provide a deeper description

of this domain in its physiological environment.

References:

1. Lu M, Lin SC, Huang Y, Kang YJ, Rich R, Lo YC, Myszka D, Han J and Wu H, Molecular cell 26(5):689-702

(2007)

2. Galbán S. and Duckett CS, Cell Death & Differentiation 17(1):54-60 (2010)

Presented by: Panagis Polykretis

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79

Sticking together: A Portrait of Protein Aggregation

Diseases

Sheena E. Radford Astbury Centre for Structural Molecular Bio logy, Univers i ty of Leeds, UK .

Understanding how different proteins assemble into the ordered, insoluble aggregates

associated with amyloid disease is a formidable challenge. Whilst it is generally accepted

that protein misfolding is required for the formation of amyloid fibrils, the point at which the

folding and aggregation free energy landscapes diverge, and how or why non-native states

of proteins are able to aggregate aberrantly, remain obscure. Even more challenging is the

identification of early aggregation-prone monomers and oligomeric species and their

structural characterisation, since such species are aggregation-prone, short-lived and

rapidly equilibrating. In this seminar I will describe how we have been using different

biochemical and biophysical methods to reveal insights into the mechanism(s) by which

normally soluble proteins convert into amyloidogenic conformations, how bimolecular

collisions between non-native proteins can result in very different outcomes of assembly,

and how we have used small molecules to modulate the aggregation process.

Presented by: Sheena E. Radford

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80

Dynamic Structural Interactions and Function of Membrane-

Bound Complexes of Cytochrome-P450 and its Redox

Partners

Meng Zhang, Thirupathi Ravula, Elke Prade, Kazutoshi Yamamoto,

Sang-Choul Im, Lucy Waskell, Ayyalusamy Ramamoorthy Biophys ics and Depar tment of Chemistry, Universi ty of Michigan, Ann Arbor MI 48109, USA

([email protected])

In spite of recent developments in structural

biology, membrane proteins continue to pose

tremendous challenges to most biophysical

techniques. High-resolution structure determination

of single-pass transmembrane proteins is even more

challenging as they exhibit dynamically disordered

structural folds that are not suitable for studies by

most biophysical techniques. My group has been

developing NMR methods and model membranes

such as nanodiscs and bicelles consisting of bulk

water that enable native structural folding of single-

pass transmembrane cytochrome proteins

(cytochrome b5, cytochrome P450 and cytochrome

P450-reductase) to measure dynamic structures, protein-protein interactions, and protein-

membrane interactions under physiological conditions. High-resolution experimental results

obtained from lipid bilayers containing full-length functional complexes of cytochrome-P450

and its redox partners will be presented along with results from solid-state (static, MAS and

DNP) and solution NMR experiments. Challenges in solving high-resolution dynamics

structures of these large protein-protein complex and obtaining biologically meaningful

atomic-level insights, benefits of nanodiscs, limitations of NMR techniques, and the role of

protein-protein interactions in the electron transfer mechanism that enable the function of

the mother nature’s blow-torch (cytochrome P450) will be discussed.

References:

1. Zhang et al, Angew. Chem. Int. Ed. Engl., 55, 4497-4499 (2016)

2. Zhang et al, J. Biol. Chem., 290, 12705-12718 (2015)

3. Zhang et al, Sci. Rep., 5, 8392 (2015)

4. Huang et al, J. Biol. Chem., 290, 4843-4855 (2015)

5. Huang et al, Biophys. J., 106, 2126-2133 (2014)

6. Ahuja et al, J. Biol. Chem., 288, 22080-22095 (2013)

7. Yamamoto et al, J. Magn. Reson., 237, 175-181 (2013)

8. Yamamoto et al, Sci. Rep., 3, 2556 (2013)

9. Yamamoto et al, Sci. Rep., 3, 2538 (2013)

10. Dürr et al, BBA Biomembranes 1768 3235-3259 (2007)

Acknowledgments: Funding support from National Institutes of Health (to A.R.)

Presented by: Ayyalusamy Ramamoorthy

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81

Investigation of STIM1OASF and Orai1 protein-protein

interactions using solution state NMR

Petr Rathnera, Christoph Romanin

b, Norbert Müller

a

a Ins t. o f Organic Chemistry , Johannes Kepler Univers ity of L inz, Altenberger Straße 69,

4040, L inz, Austr ia

(petr [email protected]) b

Ins t. o f Biophys ics, Johannes Kepler University of L inz, Gru bers traße 40, 4020, L inz,

Austr ia

The stromal interaction molecule (STIM1) and Orai1 channel are main constituents of the

calcium release-activated calcium current channel (CRAC) affecting T-cell activation and

mast cell degranulation.1 Orai1 is the protein forming the highly selective plasma membrane

calcium ion channel, which is activated by STIM1.2 The EF hand of STIM1, located in the

ER, senses the decrease of calcium concentration and responds by causing the

oligomerization of STIM1 in solution. The small Orai1 activation fragment (OASF) of STIM1

undergoes an intermolecular transition into an extended conformation, when interacting with

the Orai1 channel.4 Recently, solution state NMR spectroscopy revealed interaction sites

between part of the OASF fragment and the C-terminal domain of Orai1.5 Here we present

initial solution state NMR studies together with DLS and CD spectroscopy of the entire 257

aa OASF fragment including sample and buffer optimization in order to assess the

interaction sites and structural dynamics of the interactions these two proteins by solution

NMR.

References:

1. Liou J, Kim ML, Heo WD, et al, Current Biology 15, 1235-1241 (2005)

2. Muik M, Fahrner M, Derler L, et al, Journal of Biological Chemistry 284(13), 8421-8426 (2009)

3. Luik RM, Wang B, Prakriya M, et al, Nature 454(7203), 538-542 (2008)

4. Muik M, Fahrner M, Schindl R, et al, EMBO Journal 30(9), 1678-1689 (2011)

5. Stathopulos PB, Schindl R, Fahrner M, et al, Nature Communications 4, 2963 (2013)

Acknowledgments:

We acknowledge support by the Austrian Science Funds FWF project NanoCell (W1250) and the Austro-

Czech RERI-uasb NMR Center in Linz.

Presented by: Pert Rathner

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82

NMR for biomaterials

Enrico Raveraa, Tommaso Martelli

b, Linda Cerofolini

a-c, Vladimir K

Michaelisd-e

, Ta-Chung Ongd-f

, Eric G Keelerd, Stefano Giuntini

a-c,

Alexandra Loukaa-c

, Manuel Hafnerg, Gil Goobes

h, Marco Fragai

a-c,

Christian FW Beckerg, Ayyalusamy Ramamoorthy

i, Robert G Griffin

d,

and Claudio Luchinata-c

a

Magnet ic Resonance Center (CERM) , Univers ity of Florence and CIRMMP, I taly .

( [email protected] f i . i t ) b

Giot tobiotech S.R.L. , I ta ly.

cDepartment of Chemistry "Ugo Schif f ", Univers ity of Florence, I ta ly.

d Francis Bit ter Magnet Laboratory and Department of Chemistry , Massachusetts Inst i tu te

of Technology, United States.

e

Department of Chemistry , Univers i ty of Alberta, Canada.

f

Laboratory for Inorganic Chemistry, Depar tment of Chemi stry, ETH Zur ich, Switzer land.

g

Inst i tu te of Bio logical Chemistry , Depar tment of Chemistry , University of Vienna,

Austr ia.

h

Department of Chemistry , Bar - I lan Univers i ty, Israel .

I

Department of Chemistry and Biophys ics , Univers ity of Michigan, Uni ted States

High quality SSNMR spectra can be obtained from immobilized proteins, contributing to

the understanding of their structural features in these environments.1,2 Despite sensitivity

loss in 13C-detection based experiments due to the dilution imposed by the biomaterial

matrix, 1H detected experiments result in high resolution even in fully protonated samples.

DNP can also help in the characterization of these samples.3 The NMR properties of

immobilized enzymes will be thus analyzed for implications in 1H detection and DNP.

References:

1. Fragai M, Luchinat C, Martelli T, Ravera E, Sagi I, Solomonov I, Udi Y, Chem. Commun 50, 421 - 423

(2014)

2. Martelli T, Ravera E, Louka A, Cerofolini L, Hafner M, Fragai M, Becker CFW, Luchinat C, Chemistry–A

European Journal 22, 425-432 (2016)

3. Ravera E, Michaelis VK, Ong TC, Keeler EG, Martelli T, Fragai M, Griffin RG, Luchinat C, ChemPhysChem

16, 2751-2754 (2015)

Acknowledgments: This work has been supported by Ente Cassa di Risparmio di Firenze; by EC: Bio-NMR n.

261863, pNMR n. 317127 and COST action TD1103; the EU ESFRI Instruct through its Core Centre

CERM/CIRMMP, Italy and one R&D award; FIRC though a triennial fellowship "Guglielmina Locatello e Gino

Mazzega" (17941).

Presented by: Enrico Ravera

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83

Soluble protein complexes investigated by MAS solid-state NMR

Bernd Reif Technische Univers ität München (TUM), Department Chemie, L ichtenbergstr . 4, 85747

Garching, Germany; Helmholtz -Zentrum München (HMGU), Ingols tädter Landstr . 1 , 85764

Neuherberg, Germany. (re i [email protected])

MAS solid-state NMR techniques are applicable for the structural characterization of large

soluble protein complexes, in case the tumbling correlation time exceeds the rotor period.

Immobilization of the protein complexes is facilitated by sedimentation which is implied by

fast magic angle spinning. We show here that the approach is applicable to study the

interaction between misfolding proteins and molecular chaperones such as the small heat

shock protein (sHSP) αB-crystallin (B). We find that the resolution obtained in the proton

dimension is crucial to resolve resonances originating from the asymmetric dimer. We find

that B employs different interfaces to capture fibrillar or amorphous substrate proteins.

Presented by: Bernd Reif

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84

Order disorder transitions in the regulation of

transcription: biomedical implications

Xavier Salvatella

Ins t i tute for Research in Biomedic ine ( IRB Barcelona) , The Barcelona Inst i tu te of Sc ience

and Technology, Bald i r i Reixac 10, 08028 Barcelona, Spain and ICREA, Barcelona, Spain.

(xav ier .salvate l [email protected])

The initiation of gene expression relies on

transient protein protein interactions between the

transactivation domains of transcription factors and

the basal transcription machinery. Such

transactivation domains are in general intrinsically

disordered and their affinity for the transcription

machinery and, as a consequence, their ability to

activate transcription can be modulated by

changes in their propensity to adopt secondary

structure as well as by post translational

modifications. In my laboratory we are very interested in understanding how the transcription

factor androgen receptor activates transcription because inhibiting this process represents a

powerful therapeutic approach to halt the progression of prostate cancer that has become

refractory to hormone therapy, the first line treatment for this disease. In my talk I will

present our current understanding of this topic, which we have obtained by using nuclear

magnetic resonance as well as cell biology techniques.

Acknowledgments: This project is funded by grants awarded by Marató de TV3 (102030), MINECO (BIO2012-

31043) and the ERC (648201)

Presented by: Xavier Salvatella

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85

Protein dynamics from solid-state NMR, MD and

crystallography: from fundamental aspects to functional

motion in a large enzyme complex

Vilius Kurauskasa, Pavel Maceka, Olga N. Rogachevac, Diego F. Gautoa, Hugo

Fragaa, Sergei A. Izmailovc, Anastasya Shilovad, Peixiang Maa, Yi Xuec,

Nicolas Coquelleb, Tairan Yuwenb, Isabel Ayalaa, Audrey Hessela, Joyce

Woodhousea, Oleg Mikhailovskiib, Jérôme Boisbouviera, Jacques-Philippe

Colletiera, Nikolai R. Skrynnikovb,c, Paul Schandaa a Ins t i tut de Bio logie Structura le ( IBS) , Grenoble, France;

b Purdue Univers ity , USA;

c St Petersburg state univers ity , Russia ;

d European Synchrotron Radiat ion Faci l i ty ,

Grenoble, France

Biomolecular solid-state NMR overcomes the solubility and size limitation which are imposed to its solution-state counterpart, and it therefore allows exploring new fields of research and answer questions that are difficult to address otherwise. In this presentation we will present recent results of our protein dynamics studies, ranging from fundamental biophysical questions to mechanistic insights in difficult biological subjects.

In the first part of this presentation, we will focus on a fundamental question. How does the crystalline environment impact protein dynamics? We investigate this question by applying in parallel three different methods, solid-state NMR, MD simulations and X-ray crystallography, to different crystal forms of the same protein molecule, and investigate motions ranging from picoseconds to milliseconds. Interestingly, we find evidence that different crystals show different motions; in particular, we provide evidence for overall “rocking” motion of proteins in some crystals, and demonstrate that these motions impact resolution in NMR and crystallography, and that they may be a factor that limits X-ray data quality. Moreover, we find that a slow conformational “switch” motion, present in solution, is retained in crystals, but that its time scale, and the relative populations depend on the packing in the crystal and on inter-molecular interactions. By combining MD and NMR results, we provide evidence that rocking and intramolecular conformational exchange may be coupled and mediated by contacts to neighboring molecules. Our data thus suggest extended networks of dynamics in crystals.

In the second part we turn to a biological topic, in which dynamics is expected to play an important role. In particular, we study a large (12x39 kDa) enzymatic assembly, which forms a hollow sphere that house the active sites. Substrate entry/product exit and possible gating are open questions which cannot be entirely understood by available crystal structures. We show that assignment of such large proteins is possible (to our knowledge the largest protein assigned so far), enabling us to probe interactions and dynamics at a majority of backbone and side chain sites. Our data reveal a cluster of dynamic residues, of which we demonstrate a functional importance by biochemical experiments. We discuss possible mechanisms of gating and long-range communication in this enzyme.

Acknowledgments: This work was supported by the European Research Council (ERC Stg ProtDyn2Function

Stg 311318)

Presented by: Paul Schanda

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86

Proton-detected solid-state NMR of the 42 kDa maltose-

binding protein using 111 kHz Magic Angle Spinning

Tobias Schubeis, Loren B. Andreas, Jan Stanek and Guido

Pintacuda Ins t i tute des Sc iences Analyt iques, CRMN, 5 Rue de la Dua, 69100 Vi l leurbanne, France.

( tobias.schubeis@ens - lyon.fr )

Solid-state NMR has the potential to be a major technique to study the structure and

dynamics of proteins for which diffracting crystals are not within reach. So far the need for

large quantities of isotope labeled protein and the cumbersome analysis of moderately

resolved spectra restrained a more general application. Further improvements in sensitivity

and resolution are needed to allow a routine protein structure determination by solid state

NMR. Here we show that these challenges can be overcome by employing ultra-fast magic-

angle spinning (MAS) at rates larger than 100 kHz in small 0.7 mm rotors, at high magnetic

fields. Under these conditions, efficient detection of 1H nuclei becomes possible in fully-

protonated systems. The use of 0.7 mm rotors reduces the sample requirements to less

than 500 g without sacrificing sensitivity in the measurements. Narrow proton line-widths

result in a drastic improvement in resolution, and at the same time significantly longer

nuclear coherence lifetimes can be achieved, enabling the acquisition of multidimensional

correlation experiments. Ultrafast spinning narrows spectral resonances as well as or, for

large proteins, better than Brownian motion on which solution NMR relies, removing a

fundamental barrier to the NMR studies of large systems. This is demonstrated using a fully-

protonated microcrystalline sample of the maltose binding protein (42 kDa), a system

suitable to explore the size limits of 2D and 3D spectroscopy and develop approaches to

obtain sequences specific information in even larger proteins.

Presented by: Tobias Schubeis

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87

How dynamic RNA structure induces cellular function

Harald Schwalbe Ins t i tute for Organic Chemist ry and Chemical Bio logy, Center for Biomolecular Magnet ic

Resonance (BMRZ), Johann Wolfgang Goethe -Univers i ty Frankfur t , Max -von-Laue-Str . 7,

60438 Frankfurt .

([email protected] frankfurt .de)

RNA plays an important role in cellular regulation of gene expression. In this contribution,

we will discuss how coupling of RNA synthesis, RNA folding and ligand-induced RNA

refolding is coupled to the regulation function of RNA. Further, we show how the cellular

environment modulates RNA capacity to function as regulation switch.

Presented by: Harald Schwalbe

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88

NMR study of biosurfactants and bioemulsifiers derived

from marine organisms

Georgios A. Spyrouliasa, Konstantinos D. Marousis

a, Aikaterini

Argyrioua, Maria Birkou

a, Aikaterini Zompra

a, Konstantinos Gardikis

b

a Department of Pharmacy, Univers i ty of Patras, GR -26504, Patras, Greece.

(G.A.Spyroul [email protected] ) b

Apiv i ta S.A., Industr ia l Park of Markopoulo 19003 Markopoulo Mesogaias Greece.

Surfactants and emulsifiers (surface active agents; SAs), constitute an important class of

(bio)chemical agents that are widely used in almost every sector of modern industry1,2.

However, while having a tremendous industrial importance most of them are synthetically

manufactured using petrochemicals, which are non-renewable and also have a potentially

toxic effect on humans and the environment. On the other hand, marine bacteria can now

produce such a class of bioorganic compounds via the utilization of biological systems and.

Our group attempts to produce, separate characterize and study with NMR spectroscopy (in

vitro and in cell) a number of newly produced bio-surfactants, in order to elucidate their

structure and other physicochemical properties. Since these compounds represent a family

of reagents with a wide range of applications in food industry, cosmetics and

pharmaceuticals, attempts to optimize their production will be pursued through the

monitoring of the fermentation process using in-cell NMR (NMR-fermentanomics)3.

References:

1. Banat IM et al. Front. Microbiol. 5, 697 (2014)

2. Banat IM et al. Appl. Microbiol. 87, 427-444 (2010)

3. Bradley SA et al. J. Am. Chem. Soc. 132, 9531-9533 (2010)

Acknowledgments: This work is financially supported by H2020 “MARISURF” (nr. 635340).

Presented by: Georgios A. Spyroulias

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89

Structural features of Androgen Receptor N/C interaction

Elzbieta Szulca, Daniele Mungianu

a, Giulio Chiesa

a, Busra Topal

a,

Jesús Garcíaa, Victor Buzón

a, Xavier Salvatella

a,b

a IRB Barcelona, Spain.

b

ICERA.

Prostate cancer (PCa) is the second most common cancer and the fifth leading cause of

death from cancer in men1. It is well known that androgen receptor (AR), a steroid hormone

receptor, is important for PCa progression2. Nevertheless, in spite of AR signaling pathway

being such an important target in PC, the biology of this receptor is still not fully understood.

AR is composed of four functional domains: an N-terminal transactivation domain (NTD), a

DNA binding domain (DBD), a hinge region (H), and a ligand binding domain (LBD)3. Even

though each of its domains has a distinguished function, there is a wide body of evidence for

inter-domain communication. One of the best studied inter-domain interaction of the protein

is the N/C interaction, that has been reported to be indispensible for AR transactivation4.

The communication between the intrinsically disordered NTD and globular LBD of AR has

been proposed to facilitate coactivator recruitment by generating alternative binding sites5.

Here, I present our first steps towards understanding the consequences of this interaction

on the structure of NTD by using NMR spectroscopy and other biophysical techniques.

References:

1. http://globocan.iarc.fr

2. Wong YN et al. Nat. Rev. Clin. Oncol. (2014)

3. Claessens F et al. Nuc. R. Sig. (2008)

4. van Royen ME et al. J. Cell. Sci. (2012)

5. Claessens F. Nuc. R. Sig. (2008)

Presented by: Elzbieta Szulc

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90

Structural disorder of monomeric α-synuclein persists in

mammalian cells

Francois-Xavier Theilleta-c

, Andres Binolfia, Andrea Martorana

b,

Daniella Goldfarbb, Philipp Selenko

a

a Dpmt of Structural Bio logy, Leibniz Inst i tu te FMP -Ber l in , R. Roessle Str . 10 , 13125,

Ber l in , Germany b

Dpmt of Chemical Physics, Weizmann Inst i tute of Sc ience, Rehovot 76100, Israel c Present Address: Dpmt of Structural Bio logy, CNRS, Inst i tu te of Integrat ive Bio logy of

the Cel l , CEA-Saclay-Univers ity of Par is Saclay, 91191 Gif -sur-Yvet te, France

( francois-xav ier. thei l le [email protected] )

The native structure of Parkinson’s disease protein -

synuclein is a matter of debate. After delivering 15N-labelled -

synuclein in mammalian cells, we characterized its chemical

modifications, its conformational behavior and its interactions

with the cellular environment using both in-cell NMR and EPR

[1,2]. Hence, we have observed that -synuclein adopts the

same conformations and dynamics in cells than in vitro, and

protects its central aggregating region from cellular

interactions. However, oxidative conditions do have

irreversible impacts on -synuclein post-translational modifications, which we monitored at

the atomic scale using our recent protocols [3,4]. We will discuss it with regard to other

recent in-cell structural studies [5,6].

References:

1. Theillet FX et al, Nature 530, 45-50 (2016).

2. Binolfi A et al, Nat Commun 7, 10251 (2016)

3. Theillet FX et al, J Biomol NMR 54, 217-236 (2012)

4. Theillet FX et al, Nat Protoc 8, 1416-1432 (2013)

5. Theillet FX et al, Chem Rev 114, 6661-6714 (2014)

6. Smith MJ et al, Curr Opin Struct Biol 32, 39-47 (2015)

Presented by: Francois-Xavier Theillet

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91

The complex of cytochrome P450cam and putidaredoxin

characterized by paramagnetic NMR

Yoshitaka Hirumaa, Simon P. Skinner

a, Mathias A. S. Hass

a, Yuki

Kikuib, Wei-Min Liu

a, Betül Ölmez

a, Anneloes Blok

a, Monika

Timmera, Alexander Kloosterman

a, Hiroyasu Koteishi

b, Masaki

Nojiria,b,c

, Marcellus Ubbinka

aLeiden Ins t i tute of Chemistry, Leiden University , Einste inweg 55, 2333 CC Leiden, The

Nether lands. ([email protected] idenuniv .n l) bDepar tment of Chemistry, Osaka Univers i ty .

cRIKEN SPr ing-8 Center, Japan.

Cytochrome P450cam (P450cam) is the archetypal cyt. P450.

The structure of the complex with its electron transfer (ET) partner

putidaredoxin (Pdx) has, however, remained elusive. As the ET

steps limit the rate of catalysis and Pdx binding is required to start

the reaction, elucidating the details of the interaction is essential to

fully understand the actions of this enzyme. Paramagnetic NMR

was used to determine how Pdx docks P450cam. Using 463

distance and orientations restraints from several attached lanthanide probes (figure) a high

resolution structure was obtained (PDB code 2M56). Independent validation was provided

by solving the crystal structure of the complex solved to 2.5 Å resolution (PDB code 3W9C).

The structures of the complex in solution and in the crystal show the same orientation of

Pdx. Two ET coupling pathways are identified [1]. In the crystal state, P450cam is found in

the open state but this is attributed to a crystal packing artefact, because paramagnetic

NMR experiments show that in solution P450cam remains closed upon binding to Pdx [2].

The solution measurements further demonstrate the presence of a lowly populated

encounter state in which Pdx samples a large part of the P540cam surface [1].

References:

1. Hiruma Y et al., Ubbink M. J. Mol. Biol. 425, 4353-4356 (2013)

2. Skinner S et al., Ubbink M. PNAS 112, 9022-9027 (2015)

Presented by: Marcellus Ubbink

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92

Examining The Speciation and Structure of Metal Center

in Cells Using High-Field Electron Paramagnetic

Resonance

Leandro Tabares, Eduardo Bruch, Vincent Ching and Sun Un

Inst i tu te for Integrat ive Biology of the Cel l ( I2BC), Department of Biochemistry ,

Biophys ics and Structura l Bio logy, Univers i té Par is -Sac lay, CEA, CNRS UMR 9198, Gif -

sur-Yvette, F-91198, France. (sun.un@cea. fr )

Transition metals play critical roles in cellular physiology ranging from photosynthesis and

respiration to protection against oxidative stress and pathogenic attack. Manganese(II)

presents an interesting case. It participates in many important processes, not only as

cofactors of enzymes, but even possibly as part of small catalytic inorganic complexes, such

as those of phosphates and carbonates. It has been proposed that such Mn(II) species help

confer radio-resistance to D. radiodurans, a bacteria that can withstand greater than 1000

times more radiation than human cells. In order to understand their possible role, it is

important to first establish their speciation. Conventional metallomics approaches are not

well suited for small potentially labile complexes. We will describe how high-field EPR can

be used to not only obtain a detailed in-cell picture of Mn(II) speciation in D. radiodurans, but

also how it changes over time. This has allowed us to assess whether small complexes play

any significant role in radio-protection and managing oxidative stress. Beyond speciation

studies, recent work by others have shown that it is possible to study the in-cell structure of

proteins using electron-electron double resonance (DEER or PELDOR). We will discuss our

recent work on self-assembling metal spin labels (SAMSL) based on Mn(II) and Gd(III) as

an approach for making such nanometric distance measurements inside cells.

Presented by: Sun Un

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93

Slow protein dynamics probed by NMR: at the heart of

protein evolvability

Mariano M. González, Guillermo Bahr, Lisandro J. González and

Alejandro J.Vila Ins t i tuto de Bio logía Molecular y Celu lar de Rosar io ( IBR), CONICET – Univers idad

Nac ional de Rosar io; Área Biofís ica, Depar tamento de Química Bio lógica, Facultad de

Cienc ias Bioquímicas y Farmacéut icas, UNR, 2000 Rosar io, Argent ina ( v i la@ibr-

conicet .gov.ar )

The understanding of protein evolution depends on the ability to

relate the impact of mutations on molecular traits to fitness at the

cellular level. Biological activity and stability are key features in

shaping protein evolutionary landscapes. Instead, conformational

dynamics, has been thoroughly studied at the molecular level, but

the impact of dynamics in evolution has not been traced to the

cellular level. We have used NMR to study the intrinsic dynamic

features of a metallo-β-lactamase enzyme along a defined

evolutionary pathway in which optimization of the enzyme

performance is due to augmented dynamic features, resulting in a neat phenotype, i.e. cell

survival. We can also assess the interaction of these mutations along evolution, and identify

how some evolutionary pathways are preferred. Optimization of protein dynamics, however,

entails a loss of stability, which in some cases, asks for compensatory effects. We have also

analyzed a series of metallo-β-lactamase variants in which metal binding plays a stabilizing,

compensatory role, restricting dynamics and preventing protein degradation within the cell.

Overall, we can correlate the NMR-based description of protein dynamics at an atomistic

level to cell survival under certain conditions.

References

1. Tokuriki N, Tawfik DS, Science 324,203-7 (2009)

2. González MM, Abriata LA, Tomatis PE, Vila AJ, Mol Biol Evol. doi:10.1093/molbev/msw052 (2016)

Presented by: Alejandro J.Vila

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94

Outer membrane protein folding by the BAM complex

Michael Zahn, Leonor Morgado, Irena Burmann, Jean-Baptiste

Hartmann, Timm Maier, Sebastian Hiller Biozentrum Basel , Univers ity of Basel , Kl ingelbergstrasse 50 / 70, 4056 Basel ,

Switzer land. (michael [email protected])

β-barrel outer membrane proteins (Omps) are key functional components of the outer

membranes (OM) of Gram-negative bacteria, mitochondria and plastids. For example, they

mediate transport across the membrane, act as receptors or are involved in bacterial

pathogenicity. The biogenesis of Omps requires a protein from the Omp85 family of proteins

to mediate the insertion and translocation into the OM. In Gram-negative bacteria, this role

is fulfilled by the Omp85 protein BamA, the central protein of the BAM complex. BamA

comprises five substrate-interacting N-terminal POTRA domains and a C-terminal 16-

stranded transmembrane β-barrel domain1. Despite the recent elucidations of crystal

structures of the BAM complex2-4, the translocation and insertion mechanism of chaperone-

bound unfolded Omps into the OM remains still elusive. NMR spectroscopy allows studying

highly dynamic systems at atomic resolution and it is thus a method of choice for the

elucidation of the BamA functional mechanism. Here, we show initial results for the

preparation of BamA samples in detergent micelles, bicelles and lipid bilayer nanodiscs and

our progress towards sequence-specific resonance assignments for the BamA β-barrel

domain.

References:

1. Noinaj et al., Nature 501(7467), 385-390 (2013)

2. Gu et al., Nature 531 (7592) 64–69 (2016)

3. Han et al., Nat. Struct. Mol. Biol. 23(3), 192-196 (2016)

4. Bakelat et al., Science 351(6269), 180-186 (2016)

Presented by: Michael Zahn

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95

Sp140 SUMOylation and DNA binding

Chiara Zucchellia, Simone Tamburri

b-c, Giuseppe Filosa

d, Angela

Bachib, Giovanna Musco

a

a Biomolecular NMR Unit , San Raf faele Sc ient i f ic Ins t i tute, 20132, Mi lano, I ta ly.

(zucchel l i .ch iara@hsr. i t ) b

Funct iona l Proteomics, IFOM-Firc Inst i tu te of Molecular Oncology, 20139, Mi lano, I taly c San Raf faele Univers ità Vi ta -Salute, 20132, Mi lano, I taly

d Dip. Biotecnologie e Biosc ienze, Univers i tà degl i Studi di Mi lano -Bicocca, 20126,

Mi lano, I ta ly

Sp140 is a IFNg-inducible leukocyte-specific nuclear protein, with unknown structure and

function1,2. Since 2008 an increasing number of evidences implicate Sp140 in the etiology of

blood tumors (CLL3-6, MLL7,8). Sp140 harbours chromatin binding domains (SAND domain,

PHD finger, BRD) and indeed we proved that Sp140 binds to chromatin (IF, ChIP-seq) and

interacts with proteins involved in RNA splicing, transcription and chromatin remodeling

(interactome study by co-IP and MS).

Our structural and functional study of Sp140 domains (NMR and other biophysical and

biochemical techniques) shows that: PHD finger does not bind to histones but to the PPIase

Pin19; BRD recognizes acetylated histone H4 tail; SAND interacts with DNA. We are

currently focusing on: a) Sp140 binding to DNA: we are solving the solution NMR SAND

structure and studying both the structural determinants for DNA interaction and sequence

specificity; b) Sp140 SUMOylation: our hypothesis that PHD works as intramolecular SUMO

E3 ligase for the adjacent BRD is supported by the facts that: PHD and BRD form a

structural unit, named here “PB” (sequence alignment, NMR spectra); PB SUMOylation is

achieved through in vitro reactions; Sp140 is SUMOylated in vivo (Daudi cells) and four of

the identified SUMOylation sites are in BRD; Ubc9 (SUMO E2 ligase) binds to PHD and

BRD (NMR titrations). Using as substrate a peptide containing one of the BRD SUMOylation

sites, we are performing in vitro SUMO1 conjugation assays and rate calculation10 with and

without PHD, to prove a direct involvement of PHD in BRD SUMOylation.

References:

1. Dent AL et al. Blood 15;88(4), 1423-6 (1996)

2. Bloch DB et al. J Biol Chem 15;271(46), 29198-204 (1996)

3. Di Bernardo MC et al. Nat Genet. 40(12), 1426-35 (2008)

4. Lan Q et al. Eur J Haematol 85(6), 492-5 (2010)

5. Sillé FC et al. PloS One 7(1), e29632 (2012)

6. Quesada V et al. Nat Genet 11;44(1), 47-52 (2011)

7. Bolli N et al. Nat Commun. 5, 2997 (2014)

8. Kortüm KN et al. Ann Hematol 94(7), 1205-11 (2015)

9. Zucchelli C et al. FEBS J 281(1), 216-31 (2014)

10. Yunus AA, Lima CD, Methods Mol Biol. 497, 167-186 (2009)

Presented by: Chiara Zucchelli

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96

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97

Index

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98

Abelein 11

Adams 71

Aebersold 13

Aime 12, 37

Akopjana 76

Allain 13, 15, 40

Andersson 70

Andrałojć 14

Andreas 50, 75, 76, 86 Andrianaivomananjaona 60

Argyriou 15, 88

Ayala 85

Bachi 95

Bahr 93

Baker 32

Baldus 32, 45, 77

Banci 57, 69, 78

Barbet-Massin 16, 50

Baroni 37

Baronti 17

Batta 18

Bax 19

Becker 82

Beckmann 16

Belle 60, 64

Bellotti 50

Beltrandi 20

Benda 23

Bennati 21, 54

Bentrop 58

Berardi 22, 67

Bernhard 59

Berruyer 51

Bertarello 23, 76

Binolfi 90

Birkou 88

Blok 53, 91

Böckmann 63

Bodor 24, 34, 72

Boisbouvier 85

Bolognesi 50

Bonucci 64

Bóta 34

Bougault 61

Bruch 92

Brunori 57

Buck 25

Burmann 94

Buzón 89

Cabella 37

Cadalbert 49

Cala-De Paepe 76

Carlon 73

Case 26

Cekan 43

Cerofolini 27, 39, 53, 82 Challis 38

Chaudhari 51

Chen G. 70

Chen W.N. 71

Cheng 28

Chiarlla 57

Chiesa 89

Ching 92

Ciofi-Baffoni 69

Ciurli 64

Coles 29

Colletier 85

Colombo Serra 37

Comment 28

Coquelle 85

Corral Rodriguez 22, 67

Corti 68

Coutandin 33

Coutard 58

Crowley 30

Curnis 68

Daebel 16

Dames 31

Daniëls 32, 45, 77 Danielsson 11

Dannatt 23, 50

de Argrela Pinto 32

de Oliveira 35

de Souza 35

Degano 67

Di Matteo 57

Dorn 13, 40

Dötsch 33

Drögemüller 48

Dudás 24, 34

Duss 13

Ecsédi 24, 72

Emsley 23, 50, 51, 76 Engelke 23, 76

Enkin 54

Ernst 49

Escrig 28

Farah 35

Favaro 35

Federici 57

Feigon 36

Felli 20, 23

Filosa 95

Fiocchi 68

Fizil 18

Folkers 32 Fraga 85

Fragai 27, 39, 53, 55, 82 Fringuello Mingo 37

Gajan 51

Gallo 27

Gallo 38

García 89

Gardikis 88

Gáspári 18

Gauto 85

Gebel 33

Gerbaud 60

Ghitti 22, 67

Giorgetti 50

Giuntini 39, 82

Gmeiner 40

Gógl 24

Goldfarb 41, 90

Gonnelli 23, 27, 69

González L.J. 93

González M.M. 93

Goobes 55, 82

Graham 71

Gräslund 11

Griffin 82

Griffiths 38

Gronenborn 42

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99

Grytz 43

Guigliarelli 60, 64

Hafner 82

Hajdu 18

Haraux 60

Hartmann 94

Hass 91

Herrmann 76

Hessel 85

Hiller 63, 94

Hiruma 14, 44, 91

Hock 61

Houben 45, 77

Hsu 16

Huang 16

Huber 71

Humble 28

Huster 59, 74

Im 80

Iqbal 65

Izmailov 85

Jabar 71

Jaiswal 46

Jaudzems 70, 75, 76

Jensen 28

Jeschke 13, 40

Johansson 70

Jörnvall 70

Kaila 31

Karschin 54

Kaupp 23

Keeler 82

Kehrloesser 33

Khan 47

Kikui 91

Klein 71

Kloosterman 91

Knauer 48

Knight 50

Knott 76

Kops 44

Korsak 27

Kosol 38

Koteishi 91

Kotelovica 76

Kowalewski 47

Krauskopf 33

Kronqvist 70

Krug 59

Kumar 46

Kurauskas 85

Lakomek 49

Lalli 23, 76

Landreh 70

Le Breton 60

Le Marchand 50, 76

Lee 19

Leitner 13

Lelli 51

Lends 49

Leone 75

Lesage 51, 52, 76

Lescanne 53

Levien 54

Levine 19

Lewandowski 38

Li 25

Lichière 58

Lill 69

Liu G. 54

Liu W.M. 14, 91

Longhi 20

Louka 55, 82

Luchinat C. 14, 27, 39, 53, 55, 56, 73, 82

Luchinat E. 57, 78

Luo 31

Ma 85

Macek 85

Maier 94

Makrynitsa 58

Mance 77

Mante 59

Marko 43

Marousis 58, 88

Marques 65

Martelli 82

Martin 22

Martinho 60

Martorana 90

Marx 18

Masliah 13

Maya-Martinez 61

Mazur 63

McDermott 62

Meibom 28

Meier 49, 63

Meng 70

Michaelis 82

Mikhailovskii 85

Mileo 64

Moraes 65

Morgado 94

Mühlenhoff 69

Mulder 66

Müller 81

Mungianu 89

Musco 22, 67, 68, 95 Nardelli 68

Nasta 69

Nielsen 66

Nitsche 71

Nojiri 14, 91

Nordling 70

Ntonti 58

Nyitray 24, 72

Odelius 47

Oliverirab 65

Ölmez 91

Ong 82

Osterburg 33

Otikovs 70

Otting 71

Pálfy 24, 72

Palombo 64

Papageorgiou 58

Parigi 14, 73

Pedrote 65

Pell 23

Pelmentschikov 23

Penk 74

Penzel 49

Perrakis 44

Petzold 17

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Pica 75

Picone 75

Pierattelli 20, 23

Pilla 71

Pintacuda 23, 50, 75, 76, 86 Pinto J.R. 65

Pinto C. 77

Plane 28

Poluri 46

Polykretis 78

Prade 80

Prisner 43

Quezado 65

Quilici 22, 67, 68

Radford 79

Raikwal 46

Ramamoorthy 80, 82

Rathner 81

Ravera 14, 27, 39, 55, 73, 82 Ravotti 63

Ravula 80

Reif 16, 83

Ricagno 50

Rising 70

Roche 19

Rodriguez 25

Rogacheva 85

Romanin 81

Rösch 48

Salinas 35

Salvatella 84, 89

Sanders 23

Saviello 69

Sborghi 63

Schanda 85

Schmidt 59

Schubeis 86

Schwalbe 87

Schweimer 48

Selenko 90

Shilova 85

Sigurdsson 43

Silva 65

Simorre 61

Skinner 53, 91

Skrynnikov 85

Spantgar 69

Spiliotopoulos 67

Spyroulias 15, 58, 88

Stahlberg 63

Stanek 75, 76, 86

Stathopoulos 15

Strauß 48

Su 78

Sydor 38

Szulc 89

Tabares 92

Tamburri 95

Tars 76

Tedoldi 37

Theillet 90

Timmer 53, 91

Tkach 54

Tonon 67

Topal 89

Tsai 38

Tsika 58

Tuppi 33

Ubbink 14, 44, 53, 91 Un 92

Uzarska 69

Valentic 38

van den Brandt 28

van der Schaar 45

van der Sluis 16

van Kuppeveld 45

Vila 93

von Schroetter 15

Wacha 34

Waskell 80

Wegner 23, 76

Weiler 69

Weingarth 77

Weissenhorn 61

Werner-Allen 19

Westermeier 59

Wittwer 31

Woodhouse 85

Xue 85

Yamamoto 80

Ying 19

Yulikov 13, 40

Yuwen 85

Zahn 94

Zambelli 64

Zhang L. 25

Zhang M. 80

Zompra 88

Zucchelli 95

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Page 103:  · ribosome. For the experiment, [2H,13C,15N, 100% HN]-labeled TF-RBD (13 kDa) complexed with unlabeled 50S (1.4 MDa) was sedimented in a 1.3 mm MAS rotor and 15N-1H correlation