Institut de Biologie StructuraleGrenoble / France


A Complex Mechanism Explains How a Fluorescent Protein Changes Colour

Fluorescent proteins of the GFP family are the object of intense study due to their inherent bioluminescence, and have proved to be excellent markers for cellular imaging. In the last few years, "photoactivatable" fluorescent proteins have been developed whose fluorescence properties change as a function of their illumination conditions. These novel proteins are crucial to the new "super-resolution" fluorescence imaging methods that permit images of living cells to be obtained at nanometre resolution. One of the most popular photoactivatable proteins in nanoscopy is EosFP. This protein normally fluoresces in green, but when illuminated with violet light its fluorescence changes to red. This "photoconversion" process involves the rupture of the peptide chain next to the chromophore and formation of an enlarged conjugated system, but its mechanism has proved elusive. Starting from X-ray crystallographic structures of the green and red forms of EosFP, we have employed QC/MM simulation methods to investigate possible reaction pathways and have been able to propose a mechanism for the photoconversion. Absorption of a violet photon by the protein promotes it to its first singlet excited state. Approximately once in a thousand times, the singlet undergoes a forbidden transition to a triplet state. Once there, a proton transfer occurs that leads to a cascade of events that results in rupture of the peptide backbone, elongation of the conjugated system and red fluorescence. This work is an advance in our understanding of the function of photoactivatable fluorescent proteins, and could allow the development of variants with improved photophysical properties.

Photoconversion of the Fluorescent Protein EosFP: A Hybrid Potential Simulation Study Reveals Intersystem Crossings. Micka l Lelimousin, Virgile Adam, G. Ulrich Nienhaus, Dominique Bourgeois and Martin J. Field. JACS;131(46):16814-23.

The “gagophilic” side of chemokines

The ability of cells to migrate directionally, a process known as chemotaxis, is fundamental in the context of inflammation, as in many other physio/pathological processes. In the body, navigating cells are guided by a number of molecular clues amongst which chemokines, a recently identified family of small proteins, are in the front line and function as “molecular addresses”. Implicit in the realization of this function, these soluble mediators need to be specifically localized within tissues. This appears to be achieved by a group of acidic polysaccharides known as glycosaminoglycans (GAGs), ubiquitously present in the extracellular matrix and at the cell surface, to which most, if not all chemokines bind. As such they form surface concentration gradients that migrating cells can follow to reach their destinations.
Structural and biochemical characterisation of the chemokine-GAG interface open the route to the engineering of GAG mimetic able to manipulate this biological system.

The molecular basis and functional implications of chemokine interactions with heparan sulphate. Lortat-Jacob H. Curr. Opin. Struct. Biol. 19, 543-548

How fluorescent proteins blink

Fluorescent proteins from the GFP family are remarkable markers for cell imaging. Their weak photostability, however, constitutes their principal disadvantage. If one observes under the microscope a single fluorescent molecule (a fluorescent protein, or an organic dye for example), blinking can be immediately noticed: fluorescence is not constant over time, but alternates between bright and dark periods. In the case of GFP’s, the molecular and structural origin of blinking remains mysterious. Excited states reactions can generate a transient loss of fluorescence, such as intersystem crossing to the triplet state, chromophore protonation, or chromophore isomerization. Another possibility consists in photo-induced electron transfer, which results in the production of a radical species that is unstable and nonfluorescent. In this work, we have provided evidence for such a radical species, which was generated by x-rays from the ESRF. By combining crystallography, Raman spectroscopy, and absorption and fluorescence spectroscopy, we could show that the radical state is characterized by a severe distortion of the chromophore, which accounts for the loss of fluorescence. This is the first study showing a fluorescent protein in a transiently off state. This study could allow the development of more photostable variants. The work also highlights the importance of electron transfer reactions in fluorescent proteins.

Structural Basis of X-ray-Induced Transient Photobleaching in a Photoactivatable Green Fluorescent Protein. Adam V, Carpentier P, Violot S, Lelimousin M, Darnault C, Nienhaus GU, Bourgeois D. J Am Chem Soc. ;131(50):18063-5.

A Unified Representation of Protein Structural Dynamics in Solution ?

An atomic resolution description of protein flexibility is essential for understanding the role that structural dynamics play in biological processes. While Nuclear Magnetic Resonance (NMR) spectroscopy has emerged as the method of choice for studying bio-molecular structure and dynamics in solution, NMR-based protein structure determination often ignores the presence of dynamics, representing rapidly exchanging conformational equilibria in terms of a single static structure.
In this study researchers at the IBS use the rich dynamic information encoded in experimental NMR parameters to develop a molecular and statistical mechanical characterization of proteins in solution. In contrast to previously proposed techniques, they do not restrain the conformational search, a procedure that can strongly perturb simulated dynamics in a non-predictable way, rather they gradually increase the level of conformational sampling, and identify the appropriate level of sampling via direct comparison with experimental data.
This approach presents a unified structural dynamic representation of the motional properties of proteins in solution that will provide the basis for furthering our understanding of molecular stability, folding and function, while simultaneously proposing a new methodology for the interpretation of NMR data in terms of molecular ensembles that will be applicable to a wide range of experimental systems.

Towards A Unified Representation of Protein Structural Dynamics in Solution. P.R.L. Markwick,G. Bouvignies, L. Salmon, J.A. McCammon, M. Nilges and M. Blackledge. J.Am.Chem.Soc. 131(46) : 16968-16975, 2009.

Open Days 2009

In the framework of the 2009 French science week, the IBS organized a 4-day event :
- “Proteins revealed” : From November 18 to 20, 2009, the IBS welcomed secondary school pupils
- “Perspectives on Life at the Molecular Level” : On November 21st, 2009, biologists, chemists et physicists from three different Institutes invited the lay public to discover DNA, proteins and cells.

A hundred school pupils attended a general presentation about proteins, methods used to determine their structure and also careers in research. After this talk they visited laboratories and conducted experiments.

On Saturday, November 21, a large family audience (158 people) was able to enjoy a wide range of activities

Photo credit : IBS/O.Kaikati

About fifty volunteers were mobilized to welcome secondary school pupils and the lay public and helped to make it a successful event.

An IBS team distinguished as "FRM Team 2009"

The team of Andrea Dessen (from the Membrane Protein Laboratory) has
been designated "FRM Team 2009" by the Foundation for Medical Research.
The distinction was awarded for the teams’s work on "The mechanism of
bacterial cell wall biosynthesis as a target for the development of new
Each year the program " Espoirs de la recherche " selects about twenty
research teams on the basis of particularly innovative projects that
could potentially lead to a major advance in medicine.
The award includes financial support over three years.

Putting the squeeze on sperm DNA (Nature)

Scientists discover a new way to read the histone code by studying streamlined sperm

Structure atomique de BD1In the quest for speed, olympic swimmers shave themselves or squeeze into high-tech super-suits. In the body, sperm are the only cells that swim and, as speed is crucial to fertility, have developed their own ways to become exceptionally streamlined. Scientists at the Institut de Biologie Structurale (IBS), the European Molecular Biology Laboratory (EMBL) in Heidelberg and the Institut Albert Bonniot in Grenoble, have been studying the secrets of speedy sperm. Their work, published in Nature, shows how a protein only found in developing sperm cells, Brdt, directs tight re-packaging of sperm DNA. [Presse release]

Cooperative binding of two acetylation marks on a histone tail by a single bromodomain.
Morinière, J., Rousseaux, S., Steuerwald, U., Soler-López, M., Curtet, S., Vitte, A-L., Govin, J., Gaucher, J., Sadoul, K., Hart, D.J., Krijgsveld, J., Khochbin, S., Müller, C.W. & Petosa, C.
Nature, 461(7264):664-8.

IBS researchers on Ushuaia TV (September 30)

Researchers from the IBS are featured in the first episode of an international documentary series "Architects of Change", which will run from Wednesday, September 30 on Ushuaia TV (broadcast weekly, 10 episodes).

In the first episode entitled "To innovate is to imitate," M. (Michel?) Vivaudou presents the work of his team on a new type of biosensor.

To find out more about "Architects of Change"

Design of a hydrogenase with improved oxygen resistance

The metalloprotein group of the LCCP has contributed to the design and the characterization of [NiFe] hydrogenase mutants that, in contrast to the wild type enzyme, retain catalytic activity in the presence of molecular oxygen. This may open the way to applications in which these enzymes are used for the clean production of molecular hydrogen, potential major energy carrier of the future, from water and sunlight.

Introduction of methionines in the gas channel makes [NiFe] hydrogenase aero-tolerant. Dementin S, Leroux F, Cournac L, de Lacey AL, Volbeda A, Leger C, Burlat B, Martinez N, Champ S, Martin L, Sanganas O, Haumann M, Fernandez VM, Guigliarelli B, Fontecilla-Camps JC and Rousset M. Journal of the American Chemical Society (2009) 131(29): 10156-10164

Sugar and peptide combines to fight HIV

HIV normally enters cells when gp120, a protein on the surface of HIV, binds to receptors on the host cell. In this process, gp120 first binds to one receptor - CD4 - which causes the structure of the protein to change so that it can bind to a second receptor, such as CCR5 or CXCR4. Following several studies showing that this second receptor binding site also interact with heparan sulphate, Hugues Lortat-Jacob and colleagues have now made a molecule that contains a sequence of amino acids to mimic CD4 and a sequence of carbohydrates to mimic heparan sulfate to bind to both sites on gp120. This new strategy results in very effective inhibition of HIV infection in cellular assays, and so may have implications for further antiviral efforts.

A synthetic CD4-HS glycoconjugate inhibits both CCR5 and CXCR4 HIV-1 attachment and entry. Baleux F., Loureiro-Morais L., Hersant Y., Clayette P., Arenzana-Seisdedos F., Bonnaffé B. Lortat-Jacob H., Nature Chem. Biol. 5, 743-748.

Unexpected electron transfer in the mechanism of radical SAM-dependent proteins

Radical S-adenosine-L-methionine (SAM or AdoMet) proteins are involved in chemically difficult reactions including the synthesis of cofactors, the generation of protein radicals, and the maturation of complex organometallic catalytic sites. In the first and common step of the reaction, a conserved [Fe4S4] cluster donates an electron to perform the reductive cleavage of AdoMet into methionine and a reactive radical 5’dA. species. We have used protein crystallography and theoretical calculations to show that regardless whether AdoMet serves as a cofactor or a substrate, the 5’-dA. generating mechanism should be common to the radical SAM proteins studied so far, and that electron transfer is mediated by a unique Fe from the conserved [Fe4S4] cluster.

Unexpected electron transfer mechanism upon AdoMet cleavage in radical SAM proteins.
Yvain Nicolet, Patricia Amara, Jean-Marie Mouesca, and Juan C.

Structure and functional relevance of the Slit2 homodimerization domain

Slit proteins are secreted proteins that interact with- and signal through- the Roundabout (Robo) receptors, in a heparan sulphate dependant manner. They mediate a change in axonal response from attraction to repulsion, and provide important guidance cues during neuronal development. To better understand the role of Slit domains in signalling, the crystal structure of the fourth Slit2 domain (D4) was determined and its effect on chick retinal ganglion cell axons was examined. The Slit2 D4 domain forms a stable non-symmetrical dimer through highly conserved residues on its concave face. Functional studies showed that Slit2 dimerization is functionally relevant and that heparan sulphate can inhibit the collapse response of chick retinal ganglion cell growth cones to Slit2 D4

Structure and functional relevance of the Slit2 homodimerization domain.
Seiradake E, Philipsborn ACV, Henry M, Lortat-Jacob H, Jamin M, Hemrika W, Bastmeyer M, Cusack S and McCarthy AA
EMBO Reports (2009) 10(7): 736-741

Origins of life : IBS researchers provide an update on ancestral enzymes in Nature journal

Structure-function relationships of anaerobic gas-processing metalloenzymes.
Juan C. Fontecilla-Camps, Patricia Amara, Christine Cavazza, Yvain Nicolet & Anne Volbeda
Nature, 460 (7257):814-22

Podcast (Insight reviews).

A stunning mechanism explains the functionning of a red fluorescent protein.

Fluorescent proteins are invaluable fluorescent markers in cell biology. We have studied the red fluorescent protein Keima, which exhibits a huge Stokes shift of 180 nm, an extremely useful property for multicolor experiments. Combining X-ray crystallography with absorption, fluorescence and Raman spectroscopy, we have discovered a fascinating property of Keima : the higher the pH, the more protonated the chromophore becomes. This very strange property allows explaining the large Stokes shift of the protein at physiological pH.

Reverse pH-Dependence of Chromophore Protonation Explains the Large Stokes Shift of the Red Fluorescent Protein mKeima
Sebastien Violot, Philippe Carpentier, Laurent Blanchoin and Dominique Bourgeois
J. Am. Chem. Soc., 2009, 131 (30), pp 10356–10357

Studying the mechanism of protein denaturation using SANS and SAXS

Characterization of the conformational properties of denatured proteins is essential to our understanding of the molecular basis of protein folding and stability. Here we combine small angle neutron and X-ray scattering to study the interaction of urea with the protein ubiquitin. Comparing coherent intensities scattered at zero angle, and exploiting the scattering densities of H(2)O, D(2)O, ubiquitin, and urea for X-rays and neutrons, we quantitatively determine the number of urea molecules preferentially bound during unfolding of ubiquitin. We find that a pH change from 6.5 to 2.5 triggers recruitment of approximately 20 urea molecules from bulk solution per ubiquitin molecule during the unfolding process.

Quantitative modelfree analysis of urea binding to unfolded ubiquitin using a combination of small angle X-ray and neutron scattering. Gabel F, Jensen MR, Zaccaï G, Blackledge M. JACS 2009 Jul 1;131(25):8769-71

Structural Bases for the Affinity-Driven Selection of a Public TCR against a Dominant Human Cytomegalovirus Ep

In order to understand the principles that guide emergence of public T cell responses against cytomegalovirus, we have performed structural, biophysical and functional analyses of an immunodominant public T cell receptor (RA14 TCR) directed against a major HLA-A*0201-restricted HCMV antigen (pp65495-503) and selected in vivo from a diverse repertoire after chronic stimulations. The RA14 TCR interacts with the full array of available peptide residues indicating that its emergence out of an oligoclonal antigen-specific response after repeated viral stimulations is based on a high structural complementarity with the antigen. These results highlights key parameters underlying selection of a protective T cell response against HCMV infection, which remains a major health issue in patients undergoing bone marrow transplantation.

Structural Bases for the Affinity-Driven Selection of a Public TCR against a Dominant Human Cytomegalovirus Epitope.
Stéphanie Gras, Xavier Saulquin, Jean-Baptiste Reiser, Emilie Debeaupuis, Klara Echasserieau, Adrien Kissenpfennig, François Legoux, Anne Chouquet, Madalen Le Gorrec, Paul Machillot, Bérangère Neveu, Nicole Thielens, Bernard Malissen, Marc Bonneville, and Dominique Housset.
J. Immunol. 2009 183: 430-437

A project from the IBS awarded in a national competition for innovative projects

Since 1998 a French national competition organized by the French Ministry of Research and OSEO-ANVAR has identified and helped finance the creation of startup companies with innovative technological products.

The NatX-Ray project was one of the winners of the 2009 edition in the "creation/development" category.

The NatX-ray company commercialize automated systems initially developed by an IBS team for the CRG FIP beamline at ESRF, that significantly reduce the time required for the collection and analysis of X-ray diffraction data. Potential customers include synchrotron research facilities, academic laboratories and the pharmaceutical industry.

Longitudinal-relaxation-enhanced NMR experiments for the study of nucleic acids in solution

RNAs play key roles in many cellular processes, e.g. transfer of genetic information to the ribosome, translation, catalytic ribosome activity, and regulation of gene expression. This functional versatility requires distinct three-dimensional structures and local flexibility. These properties can be studied at atomic resolution by multidimensional NMR spectroscopy. Unfortunately, solution NMR studies are currently limited to relatively small nucleic acids at high concentrations. Here we introduce new longitudinal relaxation optimized NMR experiments for the characterization of RNA base pairing interactions, RNA-ligand binding, and real-time studies of conformational transitions, that yield over a 2-fold increase in sensitivity compared to conventional pulse schemes. This allows extending the applicability of NMR to larger or less concentrated RNA samples, and addressing new questions of biological relevance.

Longitudinal-relaxation-enhanced NMR experiments for the study of nucleic acids in solution.
Farjon J, Boisbouvier J, Schanda P, Pardi A, Simorre JP and Brutscher B.
Journal of the American Chemical Society (2009) 131(24): 8571-8577

Quantitative determination of protein-backbone motions

A robust procedure for the determination of protein-backbone motions on time scales of pico- to milliseconds directly from residual dipolar couplings has been developed that requires no additional scaling relative to external references. The results for ubiquitin (blue in graph: experimental N-HN order parameters) correspond closely to the amplitude, nature, and distribution of motion found in a 400 ns molecular-dynamics trajectory of ubiquitin (red).

Protein conformational flexibility from structure-free analysis of NMR dipolar couplings: quantitative and absolute determination of backbone motion in ubiquitin.Loïc Salmon, Guillaume Bouvignies, Phineus Markwick, Nils Lakomek, Scott Showalter, Da-Wei Li, Korvin Walter, Christian Griesinger, Rafael Brüschweiler, Martin Blackledge. Angew Chem Int Ed Engl. 2009;48(23):4154-7

Accurate characterization of weak macromolecular interactions by titration of NMR residual dipolar couplings

The description of the interactome represents one of key challenges remaining for structural biology. Physiologically important weak interactions, with dissociation constants above 100 kM, are remarkably common, but remain beyond the reach of most of structural biology. NMR spectroscopy is the technique of choice for studying low affinity protein-protein interactions. Residual dipolar couplings (RDCs) provide powerful constraints on intermolecular orientation in molecular complexes, but the combination of free and bound contributions to the measured RDCs seriously complicates their exploitation for weakly interacting partners. We have developed an approach for the exploitation of the unique orientational information available from RDCs in the case of weak interaction and rapid exchange between free and bound forms. The approach relies on the measurement of RDCs in different equilibrium mixtures of partially aligned free and complex forms of the proteins, and on novel analytical approaches to determine and normalize the effective level of alignment in all equilibrium mixtures. We show that this results in the simple extraction of precise RDCs for bound forms of both partner proteins. The approach is applied to determine the three dimensional structure of the weakly interacting CD2AP SH3-C:Ubiquitin complex (Kd=132±13 µM). We expect this methodology to extend the remarkable and unique ability of NMR to study weak protein–protein complexes.

Accurate characterization of weak macromolecular interactions by titration of NMR residual dipolar couplings: application to the CD2AP SH3-C:ubiquitin complex. J. L. Ortega-Roldan, M. R. Jensen, B. Brutscher, A. I. Azuaga, M. Blackledge, N. A. van Nuland. Nucleic Acids Res. (2009). 2009 May;37(9):e70

An Infrared fluorescent protein for whole-body imaging (Science)

GFP has revolutionized cell biology, but has met limited success in whole-body imaging, because hemoglobin and oxy-hemoglobin present in tissues tremendously absorb light below 650 nm, i.e. from UV to red. Above, there is a so called ‘optical window’, limited at around 900 nm by water absorption, in which fluorescence imaging on animal is achievable. A scientist from the IBS has contributed in Roger Tsien’s lab at the University of California San Diego to the development of a fluorescent protein suitable for whole-body imaging, based on the directed evolution of a truncated phytochrome from Deinoccus radiodurans. The latter binds biliverdin, a heme breakdown product which is abundant in the organism. Fluorescence Molecular Tomography imaging of mice infected by an adenovirus expressing the Infrared fluorescent protein and specifically targeting the liver has demonstrated the potential of this genetically-encoded marker.

Mammalian expression of Infrared fluorescent proteins engineered from a bacterial phytochrome.
Shu, X., Royant, A., Lin, M.Z., Aguilera, T.A., Lev-Ram, V., Steinbach, P.A. & Tsien, R.Y.
Science 324, 804-807.

CNRS 2009 Cristal awarded to Jacques Joly (LCCP)

Jacques Joly (LCCP) is the recipient of one of the CNRS "Crystals" for the year 2009. This distinction created in 1992 is given annually to a dozen engineers, technicians and administrative staff of the CNRS for their creativity and innovative contribution to research (list of winners 2009).

The true accuracy of NMR restraints measured in liquid crystalline media

A detailed study carried out at the IBS describes the experimental parameters that are necessary to overcome problems of conformational degeneracy and to obtain protein structures with improved accuracy and precision.

16-fold degeneracy of peptide plane orientations from residual dipolar couplings: Analytical treatment and implications for protein structure determination.
Hus JC, Salmon L, Bouvignies G, Lotze J, Blackledge M and Bruschweiler R
Journal of the American Chemical Society (2008) 130(47): 15927-15937

Gergely Katona winner of the ESRF Young scientist Award 2009

Gergely Katona received the prize for his innovative experiments in the field of structural dynamics of proteins by using synchrotron radiation. The jury awarding the prize acknowledged the combination of X-ray crystallography, time-resolved Laue diffraction, time resolved wide angle X-ray scattering and Raman spectroscopy in his research, a method developed when he was part of the Laboratoire de Cristallographie et Cristallogénèse des Protéines (IBS/LCCP).

A New Method To Tackle Structural And Dynamics Change in Molecular NanoMachines

Recently IBS researchers have developed a new technique to reduce the time required to probe large biomolecular using Nuclear Magnetic Resonance spectroscopy (NMR). The experimental time has been reduced from minutes or hours to seconds time scale. This new method offers completely new opportunities for the study of structural and dynamic changes occurring in molecular nanomachines while they perform their biological function in vitro. This technique may also prove useful for real-time investigation of macromolecular folding and self-assembly.

Fast Two-Dimensional NMR Spectroscopy of High Molecular Weight Protein Assemblies. Carlos Amero, Paul Schanda, Asunción Durá, Isabel Ayala, Dominique Marion, Bruno Franzetti, Bernhard Brutscher & Jérôme Boisbouvier. Journal of the American Chemical Society; 131(10):3448-9.

The crystallographic structure of Light-Harvesting Complex II shows the active energy-transmitting state

Illustration of the fluorescence emission and quenching properties of the light-harvesting complex II from plant in various conditions: solubilized in detergent micelles, in the crystalline form, and as part of a protein aggregate.

Under high light conditions, plants use a non-photochemical quenching mechanism to dissipate excess excitation energy as heat in order to limit the generation of reactive oxygen species that could jeopardize survival. Until recently, it was a common assumption that this mechanism should involve a conformational change within light harvesting complex II within photosynthetic membranes. Scientists from the Max Planck Institute, Frankfurt, the University of Frankfurt and the Institut de Biologie Structurale, Grenoble, have obtained new data which allow excluding the possibility of such conformational changes as part of the quenching mechanism.

Crystal structure of plant light-harvesting complex shows the active, energy-transmitting state.
Tiago Barros, Antoine Royant, Jörg Standfuss, Andreas Dreuw & Werner Kühlbrandt
The EMBO Journal;28(3):298-306.

IBS at the Days of the CEA-Grenoble

On the 23rd and 24th January 2009, the IBS participated at a symposium organized by the CEA-Grenoble, the aim of which was to present the current and future activities of the CEA-Grenoble within the framework of the projects "Scientific Peninsula" and "Operation Campus".

On the 23rd a General Assembly was hold for personnel of the CEA and its partners, whereas the 24th was devoted to an exhibition describing the Scientific Peninsula project and illustrating the activities of the CEA’s Institutes. The exhibition was open between 10 to 18h for CEA personnel and their families and had stands devoted to both fundamental and applied research. The IBS presented a stand in the zone dedicated to fundamental research.

Crédit photo : CEA/P.Avavian et O.Kaikati

French Structural Biology Day

Lyon-Gerland, January 15th, 2009

The purpose of the meeting is to take stock of the current state of structural biology in France in terms of ongoing projects, positioning within the wider European context, and long-term vision. Program.

Organizing committee :
- Eva Pebay-Peyroula
- Yves Bourne
- Dino Moras
- Stephen Cusak
- Félix Rey