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14th
Chianti Workshop
Magnetic Resonance for Cellular Structural Biology
Book of Abstracts
5-10 June 2016, Principina Terra (Grosseto), Italy
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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!
6
Organizing Institutions
CERM
University of Florence
Fondazione Luigi Sacconi
CIRMMP
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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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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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 .
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
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
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
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
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
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]
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
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
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
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
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
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
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
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
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
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
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
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
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)
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
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
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
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
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
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
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.
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
96
97
Index
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
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
100
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
101