High Field NMR:Sixty Years of Cost Effective Solutions to Real Problems Across Disciplines.

Hz

October 2009, prepared by the project leaders of the CNRS TGE RMN

“In the past increased magnetic fields have always led to new, often unexpected, domains of application for NMR”

PreambleThe national multidisciplinary delocalised TGE/TGIR for high-field NMR provides the environ-ment necessary for the efficient development of state-of-the-art NMR and its application to the resolution of important materials, biological and medical problems. The TGE is today comprised of six sites: Bordeaux, Gif-sur-Yvette, Grenoble, Lyon, Lille, Orléans, and gives access to a unique range of equipment including high resolution spec-trometers operating at multiple fields up to 1 GHz, and supported by technical expertise, and research groups dedicated to the development and applica-tion of novel, state-of-the-art spectroscopic and computational methodology in NMR. This infra-structure, equipped with the highest available fields, offers a unique en-vironment to the scien-t i f i c c o m m u n i t y i n Europe for the study of diverse problems in bio-logical, chemical, physi-cal, and medical sciences by NMR. The centers making up the TGE also propose an extensive doc-

toral and postdoctoral training program, and con-tribute to the formation of a new generation of spectroscopists with a broad interdisciplinary knowledge of diverse aspects of biological or mate-rials NMR. In the first year of activity, the infra-structure has given access to more than 75 projects from more than 50 different national Laboratories.

A Brief history of NMRSince its discovery in 1945, NMR has experienced astonishing technical development, motivated by the wide range of problems that it can be used to address, ranging from physics to medicine. For ex-ample, it is currently the only technique capable of determining protein structures in solution, which

immediately high-lights the strategic im-portance of this kind of spectroscopy today. Its application is how-ever by no means lim-ited to structural biol-ogy, as it can be used to study molecular systems relevant to agriculture (e.g. pesti-cides), chemical and materials problems

1

three-dimensional structures of proteins in solution

In 1986, using NMR, the group led by Wütrich determined a protein structure in solution for the first time. In 2000 he was the first to determine the structure of a

human prion protein. In 2002 he wins the Nobel Prize for Chemistry.

(zeolites, polymers, liquid crystals, pharmaceuti-cals, cosmetics….), medical diagnostics, or nano-technology, and is even relevant to oil exploration.

In fact the potential applications of NMR spectros-copy are currently principally limited by the costs associated with making available sufficiently high magnetic fields.

Over the years has progressively evolved from a curiosity driven experiment as a demonstration of fundamental aspects of the newly introduced quan-tum theory, into a cornerstone technique for the characterization of an impressively broad range of materials. Today NMR spectroscopy is a central tool in the atomic or molecular level understanding of systems as diverse as metal surfaces, catalysts, polymers, superconductors, glasses, liquid crystals, synthetic intermediates, supramolecular systems, natural products, drugs, membranes, and proteins, to name but a few. As such it has become the cen-tral analytical technique, and has revolutionized our ap-proach to the synthesis of new materials, and to the determina-tion of structure and dynamics in solids and in solution.

This phenomenal progress has been driven both by the devel-opment of the NMR experi-ment itself tackled by several

research groups around the world (notably with the development of Fourier transform NMR and multi-dimensional techniques, leading to the award of the 1991 Nobel prize in chemistry to Rich-ard Ernst), as well as by techno-logical developments in probe and magnet design. Indeed the pure fact that magnet strengths have gone from about 0.9 T (or 40 MHz for protons) in the early days to 1 GHz today has been one of the principal motors for devel-opment. It has allowed us to ac-cess progressively more and more complex systems, thereby extend-

ing the domain of application of NMR (see boxes). One of the most exciting aspects of this domain is that while we are absolutely sure that new fields of application will be found at higher fields (as has

always been the case in the past), we cannot predict where exactly higher fields will have the most im-pact. The open structure of the TGE/TGIR however guarantees that it will continue to be accessible to, and play a leading role in, these new areas of appli-cation.

Research Highlights from the Sites.The sites making up the infrastructure (Bordeaux,

2

insoluble Alzheimer’s proteins determined by MAS NMR

In the 1960s, the work of Andrew, Waugh, Pines, Stejskal and Shaeffer, provides high resolution spectra from solids spinning at the magic angle. From 1994 onwards Griffin (MIT) provides in-creasingly detailed evidence for the functional mechanisms in the mem-brane proteins rhodopsin and bacteri-

orhodopsin, shining light on the primary steps in vision; in 2002 Tycko (NIH) uses MAS NMR techniques to provide the first struc-ture of the plaque forming amyloid proteins responsible for Alz-heimer’s disease; and in 2006 Baldus (Gottingen) shows prelimi-nary three-dimensional structures for membrane incorporated pro-teins obtained from high-filed NMR spectra.

“With increasing magnetic fields NMR will continue to engender new, high impact, areas of applica-tions in the future”

magnetic resonance imaging: a clinical tool for diagnosis.

In 1973 Paul Lauterbur uses a high-resolution NMR spectrometer to provide the first Magnetic Reso-nance Image, of two test tubes filled with water. In 2006 this has a become a multi-billion dollar indus-try, and is the technique of choice for the diagnosis of many common tumors. In 2003 Lauterbur and Mansfield win the Nobel Prize in Medecine.

Gif-sur-Yvette, Grenoble, Lyon, Lille, Orléans) are all specialized in developing the NMR technique it-self. The group leaders are all well established figures in the interna-tional NMR community. Much of their work is related to providing the technical and methodological developments at the heart of Nu-clear Magnetic Resonance that al-low other research groups to make breakthroughs in applications prob-lems. Nevertheless, they have all made recent contributions them-selves to applications, with high impact discoveries. These applica-tions areas cover a very wide range, which is one of the most important features of this distributed infra-structure. The network provides services to users in areas ranging from medical science to physics. Some examples follow:

The Lyon group, working with sci-entists at MIT and CPE-Lyon, re-cently observed intermediates in surface supported metathesis catalysis that prove the mechanism for this industrially vital reaction. They also provided the experimental characterization supporting the capability of a single isolated tantalum atom on a surface to cleave molecular nitrogen. In different work, with IBCP-Lyon, they showed for first time that microcrystalline samples allow NMR to probe the details of the water-protein interactions that stabilize protein structures and control folding and unfolding processes. In yet another area, the Lyon group showed how the model animal C elegans could be successfully used as a platform to study functional genetics by NMR in connection with disease.

The Grenoble group has recently shown that slow movements along the backbone in a model protein are correlated and form a long

range network of motions, leading to the remarkable obser-vation of a standing wave ex-tending across a beta sheet. Slow motions are related to processes such as signal trans-duction and allosteric regula-tion. The group is developing innovative methods combining spectroscopic, computational, and stable isotope labeling ap-proaches for the study of mo-lecular systems of increasing size and complexity, of short-lived molecules, and of intrinsi-cally unstructured proteins. A particular focus is on the devel-opment of fast multidimensional NMR methods which will allow the study of transient structures during real time protein folding or other non-equilibrium mo-lecular processes. Highlights include structural and interac-tion studies on proteins and non-coding RNAs involved in the

process of viral replication, especially those of hu-man immunodeficiency virus (HIV), hepatitis C (HCV) and influenza viruses. Another focus is the investigation of proteins involved in bacterial cell wall synthesis that represent the most important targets of actual antibiotics. This work was recently pushed further by exploring the capability of solid state NMR in order to directly study the bacterial cell wall and to screen for interacting proteins.

3

new frontiers in physics: from su-perconductivity to quantum comput-ing

In 1945 Bloch and Purcell demonstrate the NMR phe-nomen to vali-date the emerging quantum the-

ory. They speculate it could be a useful method for calibrating magnetic fields. They win the Nobel prize for Physics in 1952. Slichter later uses NMR to provide the first experimen-tal proof of the BCS theory for superconductivity. In 1997 Gershenfeld and Chuang show that high-resolution NMR can provide the support for multi-bit quantum computation. In 2001 NMR provides the first experi-mental demonstration of the solution to Shror’s Algorithm.

metabolism, diagnosis, and personalised healthcare.

Urine was one of the first complex fluids to be studied by NMR. This led to the emergence of “metabolomics by NMR.” In the 1990s NMR spec-tra are used to determine types of cancer. In 2006 Nicholson and coworkers present results from worldwide epidemiological studies, involving thousands of subjects, determining environmental factors affecting the occurrence of diabetes and high blood pressure in whole populations. This type of NMR is playing a key role in the emer-gence of the idea of personalized health care.

The Gif-sur-Yvette group has a long term interest in natively unfolded proteins. They developed new models for the analysis of solution dynamics in proteins and elucidated the mechanisms for their folding upon interaction with biological partners in biologically relevant examples (actin monomer se-questering…). They also worked on the intimate relationships between protein primary sequences, dynamic properties and folding, with potential ap-plications in protein engineering. The structure de-termination of biomacromolecules (proteins, DNAs, ARNs…) and the analysis of their com-plexes with other biomacromlecules and ligands provided important clues towards the understand-ing of their biological functions,, that open new routes towards the development of important therapeutic agents (anti HIV molecules, antibiot-ics…).

The Orléans group has developed a world wide unique laser heating device that allows the investi-gating of structure and dynamics in the molten state by NMR up to more that 2000°C, now ex-tending to in-situ measurement of diffusion coeffi-cients. They have also develop new methods for the characterization of medium range order in glasses with experimental results that demonstrate unexpected structural details and add new con-

straints to models. Recent developments now allow characterization of "molecular motifs" in glasses allowing to sort out chemical and geometrical dis-order at the nanometer scale. They have partici-pated in the characterization of hybrid new materi-als with specific properties and applications in nanomaterials, biocompatible materials or drug de-livery. Most of these results are obtained in the course of national and international collaborations.

The Bordeaux group, with scientists at UCSD and the Burnham Institute in San Diego, succeeded in reincorporating the Pf1 membrane protein into biomembranes that are macroscopically oriented by magnetic fields (biphenyl bicelles), and determined the topology of the helical protein in the membrane using nitrogen-proton solid state NMR. In other work, in collaboration with Cancer Research UK in London, the fluidity of the nuclear envelope poles that are involved in male/female cell fusion during reproduction were measured by deuterium solid state NMR of live cells. Recently in collaboration with an INSERM team in Strasbourg who discov-ered a membranous peptide capable of inhibiting the development of plasmodium falciparum (ma-laria), the Bordeaux group was able to propose a mechanism of action by “molecular electropora-tion” as inferred from solid sate NMR, molecular modeling and electrophysiology.

4

glasses, new materials, and nanosciences

Quadrupolar nuclei have always played a leading role in NMR. Since the 90s oxygen and alumi-num NMR studies have continuously contributed to change the under-

standing we have of the structure and dynamics of glass forming materials and their related mol-ten state. This is now changing the whole way we think about the formation and structure of disordered materials. In 2006 Grey and co-workers use understanding from NMR observa-tions directly to improve the charging rate ca-pacity of lithium nickel magnanese oxide in re-chargeable batteries.

basic chemistry and catalysis

The first spectra from catalysts are recorded in the 1970s, as NMR revolutionizes the way chemists approach multi-step synthesis. In 2006 Schrock wins the Nobel Prize in Chem-istry for his development of meta-thesis, which has become central to basic industrial chemistry. In the same year he uses high-field solid-state NMR to validate the mecha-nism of olefin meta-thesis on a supported catalyst.

10Å 10Å

SiO2 H E T E R O G E N E O U S

The Lille groups develop com-plementary approaches in liquid and solid-state NMR. The two main groups are working on bio-logical applications (structural analysis of proteins) and on the development of solid-state NMR methods and their applications to inorganic materials. Since 1995, they have introduced many new developments for quadrupolar nu-clei concerning both the direct characterization of quadrupolar nuclei and the analysis of through-space or through-bond connectivities with other nuclei. These methods are applied to the development of high-field 17O NMR characterization of inorganic glasses and ceramics (e.g. sealing glasses for SOFC, antioxidation phosphate coatings). The bio-logical NMR group has focused on NMR of het-erogeneous systems, and on proteins involved in the cell cycle. The group has studied in detail the neuronal Tau protein which, upon aggregation, is one of the molecular hallmarks of Alzheimer’s dis-ease, and has used both solution and High Resolu-tion Magic Angle Spinning NMR to study the soluble and aggregated form of the protein.

These groups already have a history of collabora-tion. Work between Lyon and Grenoble recently resulted in the first quantitative analysis of internal dynamics in a solid protein, and a thesis student is currently under joint direction. Moreover, the Lyon and Grenoble sites already jointly operate a Euro-pean Large Scale Facility, in the context of an Inte-grated Infrastructure Initiative (www.ralf-nmr.fr).

Recent collaboration between Lyon and Orléans has led to the development of sophisticated meth-ods to study the details of structures in complex inorganic materials, including glasses, including very advanced ideas about efficient quantum trans-port in adiabatic processes that was highlighted in press releases around the world.

Collaboration between Orléans and Lille has re-cently resulted in three cornerstone publications describing (i) a new probe to study chemical bond-ing differences in alumino-phosphate materials, and (ii) a new method to analyze in high-resolution the connectivities of inorganic fluoride samples.

Frontier domains in NMR: New oppor-tunities at high magnetic fields for the TGE/TGIR network.Today we can identify several domains where the increased availability of high-field NMR is likely to have considerable impact in the medium term.

Obviously, the motor for high-field NMR science will continue for some years to be structural biol-ogy, as it has been for the last 15 years. The

5

solving the DNA recognition puzzle

NMR spectra are first obtained from DNA and RNA oligomers in the early 1970s. In 2004, Kaptein uses 900 MHz NMR of protein-DNA complexes to determine the kinetics and structural changes that al-low proteins to find their recognition sites in extended DNA se-quences.

from penicillin to taxol: stereochemistry in the drug industryIn 1959 Karplus proposes a dependence of H-H coupling constants on di-hedral angles. Today this forms the basis for the determination of the stereochemistry of many of the therapeutic drugs on the market, crucial to both their safety and efficiency. Recent developments combining cryo-cooled probes and high magnetic fields, have made possible the monitor-ing of enantiomeric purity by NMR of deuterium at natural abundance, us-ing liquid crystalline solvents. This allows the discrimination between enan-tiomeric forms of compounds which previously could not be resolved.

N

N

O H

S

O O-

O

HH

TROSY effect, allowing access to ever larger bio-logical molecules in solution, is predicted to be at its best at an NMR frequency close to 1 GHz. This will allow solution-state NMR studies of larger proteins, and notably allows for the possibility of

studying membrane proteins in detergent formula-tions. Also, the study of proteins in the solid state, whether micro-crystalline, fibril forming, or mem-brane incorporated, will be increasingly enabled by the increased sensitivity of higher fields. Greater accesibilty for NMR studies in such samples will provide better understanding of the mechanism of diseases, and yield new perspectives for therapy.

The whole field of nanotechnology and new mate-rials will clearly benefit considerably from increas-ing magnetic field strengths. The probe nuclei in these materials are often quadrupolar in nature. The simplifying effect of high field is absolutely spec-tacular in these cases, and should allow access to understanding the molecular level organization and properties of increasingly complex materials. This is particularly exciting as it opens up a tool which will actively aid the development of many new, high technology, materials.

Finally, one of the most exciting areas where higher fields will have great impact in the long term is that of basic analytical sciences in general, and development of analytical methods for medical diagnosis in particular. Here the principle drawback of NMR is sensitivity. This of great importance when considering the analysis of environmental samples, for example, often only available in trace quantities. The recent development of microcoil technology, and “lab on a chip” approaches, com-bined with high fields will push back the detection limits, making it possible to analyze increasingly smaller quantities, with increasing reliability.

Clearly one of the most interesting analytical ob-jectives would be to provide diagnostic and prog-nostic tools for medical applications through the analysis of biological fluids, such as urine or plasma, or biopsy type materials. There has been some very impressive progress made in this area over the last ten years, and it is clear that increased sensitivity will lead to the detection of metabolites present at lower and lower concentrations, provid-ing reliable markers for diverse diseases.

In conclusion, as in the past, it is clear that NMR will continue to provide the key to many high-impact problems in multi-disciplinary science in the future, driven forward to a large degree by the inexorable increase in magnetic field strengths.

Selected Key References for the Sub-jects Highlighted in the Boxes.L.C. Hebel and C.P. Slichter. "Nuclear Spin Relaxation in Normal and Superconducting Aluminum." Physical Review 1959;113:1504-19.

M. Karplus. "Contact Electron-Spin Coupling of Nuclear Magnetic Moments." Journal of Chemical Physics 1959;30:11.

M. Karplus. "Vicinal Proton Coupling in Nuclear Magnetic Reso-nance." Journal of the American Chemical Society 1963;85:2870.

P.C. Lauterbur. "Image Formation by Induced Local Interactions - Examples Employing Nuclear Magnetic-Resonance." Nature 1973;242:190-1.

P. Mansfield. "Multi-Planar Image-Formation Using NMR Spin Ech-oes." Journal of Physics C-Solid State Physics 1977;10:L55-L8.

M.P. Williamson, T.F. Havel and K. Wuthrich. "Solution Conformation of Proteinase Inhibitor-Iia from Bull Seminal Plasma by H-1 Nuclear Magnetic-Resonance and Distance Geometry." Journal of Molecular Biology 1985;182:295-315.

S.E. Barrett, D.J. Durand, C.H. Pennington, C.P. Slichter, T.A. Fried-mann, J.P. Rice and D.M. Ginsberg. "Cu-63 Knight-Shifts in the Su-perconducting State of Yba2cu3o7-Delta(Tc=90-K)." Physical Review B 1990;41:6283-96.

I. Farnan, P.J. Grandinetti, J.H. Baltisberger, J.F. Stebbins, U. Wer-ner, M.A. Eastman and A. Pines. "Quantification of the Disorder in Network-Modified Silicate-Glasses." Nature 1992;358:31-5.

B.T. Poe, P.F. McMillan, B. Coté, D. Massiot and J.P. Coutures “Mag-nesium And Calcium Liquids: In situ High-Temperature 27Al NMR Spectroscopy,” Science 1993;259:768-88.

A. Meddour, I. Canet, A. Loewenstein, J.M. Pechine and J. Courtieu. "Observation of Enantiomers, Chiral by Virtue of Isotopic-Substitution, through Deuterium NMR in a Polypeptide Liquid-Crystal." Journal of the American Chemical Society 1994;116:9652.

J.G. Hu, R.G. Griffin and J. Herzfeld. "Synergy in the Spectral Tuning of Retinal Pigments - Complete Accounting of the Opsin Shift in Bac-teriorhodopsin." Proceedings of the National Academy of Sciences of the United States of America 1994;91:8880-4.

N.A. Gershenfeld and I.L. Chuang. "Bulk spin-resonance quantum computation." Science 1997;275:350-6.

J.G.G. Hu, R.G. Griffin and J. Herzfeld. "Interactions between the protonated Schiff base and its counterion in the photointermediates of bacteriorhodopsin." Journal of the American Chemical Society 1997;119:9495-8.

J.F. Stebbins and Z. Xu. "NMR evidence for excess non-bridging oxy-gen in an aluminosilicate glass." Nature 1997;390:60-2.

6

“ The open structure of the TGE/TGIR guarantees that it will be ac-cessible to, and play a leading role in, these new areas of application.”

R. Zahn, A.Z. Liu, T. Luhrs, R. Riek, C. von Schroetter, F.L. Garcia, M. Billeter, L. Calzolai, G. Wider and K. Wuthrich. "NMR solution struc-ture of the human prion protein." Proceedings of the National Acad-emy of Sciences of the United States of America 2000;97:145-50.

L.M.K. Vandersypen, M. Steffen, G. Breyta, C.S. Yannoni, M.H. Sher-wood and I.L. Chuang. "Experimental realization of Shor's quantum factoring algorithm using nuclear magnetic resonance." Nature 2001;414:883.

J.T. Brindle, H. Antti, E. Holmes, G. Tranter, J.K. Nicholson, H.W.L. Bethell, S. Clarke, P.M. Schofield, E. McKilligin, D.E. Mosedale and D.J. Grainger. "Rapid and noninvasive diagnosis of the presence and severity of coronary heart disease using H-1-NMR-based metabo-nomics." Nature Medicine 2002;8:1439-44.

J.C. Lansing, M. Hohwy, C.P. Jaroniec, A.F.L. Creemers, J. Lugten-burg, J. Herzfeld and R.G. Griffin. "Chromophore distortions in the bacteriorhodopsin photocycle: Evolution of the H-C14-C15-H dihedral angle measured by solid-state NMR." Biochemistry 2002;41:431-8.

J.K. Nicholson, J. Connelly, J.C. Lindon and E. Holmes. "Metabonom-ics: a platform for studying drug toxicity and gene function." Nature Reviews Drug Discovery 2002;1:153-61.

A.T. Petkova, Y. Ishii, J.J. Balbach, O.N. Antzutkin, R.D. Leapman, F. Delaglio and R. Tycko. "A structural model for Alzheimer's beta-amyloid fibrils based on experimental constraints from solid state NMR." Proceedings of the National Academy of Sciences of the United States of America 2002;99:16742-7.

C.G. Kalodimos, N. Biris, A. Bonvin, M.M. Levandoski, M. Guen-nuegues, R. Boelens and R. Kaptein. "Structure and flexibility adapta-tion in nonspecific and specific protein-DNA complexes." Science 2004;305:386-9.

M.L. Mak, V.S. Bajaj, M.K. Hornstein, M. Belenky, R.J. Temkin, R.G. Griffin and J. Herzfeld. "Chromophore torsion early in the bacteriorho-dopsin photocycle." Biophysical Journal 2005;88:506A-A.

A.T. Petkova, R.D. Leapman, Z.H. Guo, W.M. Yau, M.P. Mattson and R. Tycko. "Self-propagating, molecular-level polymorphism in Alz-heimer's beta-amyloid fibrils." Science 2005;307:262-5.

F. Blanc, C. Coperet, J. Thivolle-Cazat, J.M. Basset, A. Lesage, L. Emsley, A. Sinha and R.R. Schrock. "Surface versus molecular siloxy ligands in well-defined olefin metathesis catalysts: {(RO)(3)SiO}Mo(=NAr)(=CHtBu)(CH(2)tBu)." Angewandte Chemie 2006;45:1216-20.

K.S. Kang, Y.S. Meng, J. Breger, C.P. Grey, G. Ceder ”Electrodes with high power and high capacity for rechargeable lithium batteries, ”Sci-ence 2006; 311:977.

T.A. Clayton, J.C. Lindon, O. Cloarec, H. Antti, C. Charuel, G. Hanton, J.P. Provost, J.L. Le Net, D. Baker, R.J. Walley, J.R. Everett and J.K. Nicholson. "Pharmaco-metabonomic phenotyping and personalized drug treatment." Nature 2006;440:1073-7.

K.A. Henzler-Wildman, M. Lei, V. Thai, S.J. Kerns, M. Karplus, D. Kern, “A hierarchy of timescales in protein dynamics is linked to en-zyme catalysis,” Nature 2007; 450: 913

P.J. Sideris, U.G. Nielsen, Z.H. Gan, C.P. Grey, “Mg/Al ordering in layered double hydroxides revealed by multinuclear NMR spectros-copy,” Science 2008; 321: 113.

Selected Recent References.

Lyon

A. Lesage, L. Emsley, F. Penin and A. Böckmann, “Investigation of Dipolar-Mediated Water-Protein Interactions in Microcrystalline Crh by Solid-State NMR Spectroscopy” Journal of the American Chemical Society 2006; 128: 8246.

B. Elena, G. Pintacuda, N. Mifsud and L. Emsley, “Molecular Structure Determination in Powders by NMR Crystallography from Proton Spin Diffusion,” Journal of the American Chemical Society 2006; 128: 9555.

B.J. Blaise, J. Giacomotto, B. Elena, M.-E. Dumas, P. Toulhoat, L. Ségalat and L. Emsley “Metabotyping of Caenorhabditis elegans re-veals latent phenotypes,” Proceedings of the National Academy of Sciences of the United States of America 2007; 104: 19808.

P. Felgines Avenier, M. Taoufik, A. Lesage, A. Baudouin, A. De Mall-mann, L. Veyre, J.-M. Basset, L. Emsley, and E.A. Quadrelli, “Dinitro-gen Dissociation on an Isolated Surface by a Single Tantalum Atom,” Science 2007; 317: 1056.

F. Blanc, R. Berthoud, C. Copéret, A. Lesage, L. Emsley, R. Singh, T. Kreickmann, R.R. Schrock, “Direct observation of reaction intermedi-ates for a well-defined heterogeneous alkene metathesis catalyst,” Proceedings of the National Academy of Sciences of the United States of America 2008; 105: 12123.

Grenoble

G. Bouvignies, P. Bernado, S. Meier, K. Cho, S. Grzesiek, R. Brusch-weiler and M. Blackledge, “Identification of slow correlated motions in proteins using residual dipolar and hydrogen-bond scalar couplings.” Proceedings of the National Academy of Sciences of the United States of America 2005;102:13885-90

P. Schanda and B. Brutscher, “Very fast two-dimensional NMR spec-troscopy for real-time investigation of dynamic events in proteins on the time scale of seconds.” Journal of the American Chemical Society 2005;127:8014-5

T. Kern, S. Hediger, P. Muller, C. Giustini, B. Joris, C. Bougault, W. Vollmer and J.P. Simorre, “Toward the characterization of peptidogly-can structure and protein-peptidoglycan interactions by solid-state NMR spectroscopy.” Journal of the American Chemical Society 2008, 130: 5618.

C. Arnero, P. Schanda, M.A. Dura, I. Ayala, D. Marion, B. Franzetti, B. Brutscher, J. Boisbouvier, “Fast Two-Dimensional NMR Spectroscopy of High Molecular Weight Protein Assemblies” Journal of the Ameri-can Chemical Society 2009; 131: 3448.

L. Salmon, G.Bouvignies, P. Markwick, N. Lakomek, S. Showalter, D.W. Li, K. Walter, C. Griesinger, R. Bruschweiler and M. Blackledge, “Protein conformational flexibility from structure-free analysis of NMR dipolar couplings : quantitative and absolute determination of back-bone motion in ubiquitin,” Angewandte Chemie 2009; 48: 4154.

Bordeaux

J.G. Beck, D. Mathieu, C. Loudet, S. Buchoux, E.J. Dufourc, “Plant sterols in "rafts": a better way to regulate membrane thermal shocks” Faseb Journal, 2007; 21: 1714.

S.H. Park, C. Loudet, F.M. Marassi, E.J. Dufourc, and S.J. Opella, Solid-state NMR spectroscopy of a membrane protein in biphenyl phospholipid bicelles with the bilayer normal parallel to the magnetic field. Journal of Magnetic Resonance 2008;193: 133.

M.A. Sani, O. Keech, P. Gardeström, E.J. Dufourc, and G. Gröbner, Magic-angle phosphorus NMR of functional mitochondria: in situ monitoring of lipid response under apoptotic-like stress. Faseb Jour-nal 2009; 23: DOI:10.1096/fj.1009-134114.

M. Garnier-Lhomme, R.D. Byrne, T.M.C. Hobday, S. Gschmeissner, R. Woscholski, D.L. Poccia, E.J. Dufourc, and B. Larijani, Nuclear envelope remnants: fluid membranes enriched in sterols and poly-phosphoinositides. PLoS One 4 2009 e4255.

A. Diller, C. Loudet, F. Aussenac, G. Raffard, S. Fournier, M. Laguerre, A. Grelard, S.J. Opella, F.M. Marassi, and E.J. Dufourc, Bicelles: A natural 'molecular goniometer' for structural, dynamical and topological studies of molecules in membranes. Biochimie 2009; 91: 744-751.

Gif-sur-Yvette

F. Ochsenbein, R. Guerois, J-M. Neumann, A. Sanson, E. Guittet and C. Van Heijenoort, “Model based on a Lorentzian distribution of corre-lation times : reconsidering the interpretation of the 15N relaxation parameters in the case of unfolded proteins.” Protein Science 2002;11:957.

M. Hertzog, C. Van-Heijenoort, D. Didry, M. Gaudier, J. Coutant, B. Gigant, G. Didelot, T. Preat, M. Knossow, E. Guittet and M.F. Carlier, “The b-thymosin/WH2 domain : Structural basis for the switch from inhibition to promotion of actin assembly.” Cell 2004;117:611.

7

C. Gaudin, M-H. Mazauric, M. Traikia, E. Guittet, S. Yoshizawa and D. Fourmy, ”Structure of the RNA signal essential for translational frame-shifting in HIV-1.” Journal of Molelcular Biology, 2005;349:1024.

P. Aliprandi, C. Sizun, J. Perez, F. Mareuil, S. Caputo, J-L. Leroy, B. Odaert, S. Laalami, M. Uzan and F. Bontems, "S1 ribosomal protein functions in translation initiation and ribonuclease RegB activation are mediated by similar RNA-protein interactions" Journal of Biological Chemistry 2008; 283:13289.

D. Stratmann, C. van Heijenoort and E. Guittet, "NOEnet-Use of NOE networks for NMR resonance assignment of proteins with known 3D structure" Bioinformatics 2009; 11:474.

Lille

I. Landrieu, M. da Costa, L. De Veylder, F. Dewitte, K. Vandepoele, S. Hassan, J.M. Wieruszeski, F. Corellou, J.D. Faure, M. Van Montagu, D. Inze, G. Lippens, “NMR structure of a small CDC25 dual-specificity tyrosine-phosphatase isoform in Arabidopsis thaliana.” Proceedings of the National Academy of Sciences of the United States of America 2004;101:16391.

G. Tricot, L. Delevoye, G. Palavit, L. Montagne, “Phase identification and quantification in a devitrified glass using homo- and heteronuclear solid state NMR.” Chemical Communications 2005;5289-91.

A. Sillen, J.M. Wieruszeski, A. Ben Younes, I. Landrieu et G. Lippens, “HRMAS NMR characterization of the Paired Helical Fragments of the neuronal Tau protein.” Journal of the American Chemical Society 2005;127:10138-9.

I. Landrieu, L. Lacosse, A. Leroy, J.M. Wieruszeski, X. Trivelli, A. Sillen, N. Sibille ,H. Schwalbe, K. Saxena, T. Langer, G. Lippens, “NMR analysis of a Tau phosphorylation pattern” Journal of the American Chemical Society 2006; 128: 3575.

Z.H. Gan, J.P. Amoureux, J. Trebosc, “Proton-detected N-14 MAS NMR using homonuclear decoupled rotary resonance”Chemical Physics Letters 2007; 435: 163.

Orléans

S.Josse, C.Faucheux, A.Soueidan, G.Grimandi, D.Massiot, B.Alonso, P.Janvier, S.Laïb, O.Gauthier, G.Daculsi, J.Guicheux, B.Bujoli, J.-M.Bouler, “Chemically Modified Calcium Phosphates as Novel Ma-terials for Bisphosphonate Delivery.” Advanced Materials 2004;16:1423-27.

M. Deschamps, F. Fayon, V. Montouillout, D. Massiot, “Through-bond homonuclear correlation experiments in Solid-state NMR applied to quadrupolar nuclei in Al-O-P-O-Al chains.” Chemical Communications 2006:1924-5.

C. Martineau, F. Fayon, C. Legein, J.Y. Buzaré, G. Silly, D. Massiot, “Accurate Heteronuclear J-Coupling Measurements in Dilute Spin Systems using the multiple-quantum filtered J-resolved experiment,” Chemical Communications 2007; 2720.

D. Laurencin, C. Gervais, A. Wong, C. Coelho, F. Mauri, D. Massiot, M.E. Smith, C. Bonhomme, “Implementation of high resolution 43Ca solid state NMR spectroscopy: towards the elucidation of calcium sites in biological materials,” Journal of the American Chemical Soci-ety 2009; 131: 13430.

G. Arrachart, G. Creff, H. Wadepohl, C. Blanc, C. Bonhomme, F. Ba-bonneau, B. Alonso, J.L. Bantignies, C. Carcel, J.J.E. Moreau, P. Dieudonné, J.L. Sauvajol, D. Massiot, M. Wong Chi Man, “Nanostruc-turing of hybrid silicas through self-recognition process,” Chemistry, A European Journal 2009; 15: 5002.

References to Collaborative Papers Be-tween the Sites.

Orléans/Lyon

M. Deschamps, D. Massiot, G. Kervern, G. Pintacuda, L. Emsley and P.J. Grandinetti, “Superadiabaticity in Magnetic Resonance,” J. Chem. Phys. 2008;127: 204110.

F. Fayon, C.Roiland, L.Emsley, D.Massiot. “Triple-quantum correlation NMR experiments in solids using J-couplings,” Journal of Magnetic

Resonance 2006;179:50-8.

F. Fayon, D. Massiot, M.H. Levitt, J.J. Titman, D.H. Gregory, L. Duma, L. Emsley, S.P. Brown. “Through-space contributions to two-dimensional double-quantum J correlation NMR spectra of magic-angle-spinning solids,” Journal of Chemical Physics 2005;122:194313.

F. Fayon, G. Le Saout, L. Emsley, D. Massiot. “Through-Bond Phosphorus-Phosphorus Connectivities in Crystalline and Disordered Phosphates by Solid-State NMR,” Chemical Communications 2002;1702-3

Lille/Orléans

Q. Wang, B. Hu, F. Fayon, J. Trébosc, C. Legein, O. Lafon, F. Deng, J.P. Amoureux, "Double-quantum 19F-19F dipolar recoupling at ultra-fast magic angle spinning NMR: application to the assignment of 19F NMR spectra of inorganic fluorides"; Phys. Chem. Chem. Phys 2009; DOI: 10.1039/b914468d.

B. Hu, J.P. Amoureux, J. Trebosc, M. Deschamps, G. Tricot, “Solid-state NMR covariance of HOMCOR spectra,” Journal of Chemical Physics 2008; 128: 134502.

J.P. Amoureux, J. Trebosc, J.W. Wiench, D. Massiot, M. Pruski. “Measurement of J Couplings between Spin-½ and Quadrupolar Nu-clei by Frequency Selective Solid State NMR,” Solid State NMR 2005:27;228-32.

D. Massiot, F. Fayon, B. Alonso, J. Trebosc, J.P. Amoureux. “Chemical bonding differences evidenced from J coupling in solid state NMR experiments involving quadrupolar nuclei,” Journal of Magnetic Reso-nance 2003;164:165-70.

Lyon/Grenoble

J. Sein, N. Giraud, M. Blackledge and L. Emsley, “The Role of 15N CSA and CSA/Dipole Cross Correlation in 15N Relaxation in Solid Proteins,” J. Magn. Reson. 2007; 186: 26.

N. Giraud, J. Sein, G. Pintacuda, A. Böckmann, A. Lesage, M. Black-ledge and L. Emsley, “Observation of Heteronuclear Overhauser Ef-fects Confirms the 15N-1H Dipolar Relaxation Mechanism in a Crys-talline Protein,” J. Am. Chem. Soc. 2006; 128: 12398.

N. Giraud, M. Blackledge, M. Goldman, A. Bockmann, A. Lesage, F. Penin and L. Emsley. "Quantitative analysis of backbone dynamics in a crystalline protein from nitrogen-15 spin-lattice relaxation." Journal of the American Chemical Society 2005;127:18190.

N. Giraud, A. Bockmann, A. Lesage, F. Penin, M. Blackledge and L. Emsley. "Site-specific backbone dynamics from a crystalline protein by solid-state NMR spectroscopy." Journal of the American Chemical Society 2004;126:11422.

M. Juy, F. Penin, A. Favier, A. Galinier, R. Montserret, R. Haser, J. Deutscher and A. Bockmann. "Dimerization of Crh by reversible 3D domain swapping induces structural adjustments to its monomeric homologue Hpr." Journal of Molecular Biology 2003;332:767.

A. Favier, B. Brutscher, M. Blackledge, A. Galinier, J. Deutscher, F. Penin and D. Marion. "Solution structure and dynamics of Crh, the Bacillus subtilis catabolite repression HPr." Journal of Molecular Biol-ogy 2002;317:131.

F. Penin, A. Favier, R. Montserret, B. Brutscher, J. Deutscher, D. Mar-ion and A. Galinier. "Evidence for a dimerisation state of the Bacillus subtilis catabolite repression HPr-like protein, Crh." Journal of Molecu-lar Microbiology and Biotechnology 2001;3:429.

A. Lesage, F. Penin, C. Geourjon, D. Marion and M. vanderRest. "Trimeric assembly and three-dimensional structure model of the FACIT collagen COL1-NC1 junction from CD and NMR analysis." Biochemistry 1996;35:9647.

For further information, contact Lyon: lyndon.emsley@ens-lyon.fOrléans: massiot@cnrs-orleans.frGif-sur-Yvette: guittet@icsn.cnrs-gif.frGrenoble: jean-pierre.simorre@ibs.frLille: guy.lippens@ibl.frBordeaux: e.dufourc@iecb.u-bordeaux.fr

8