Institut de Biologie StructuraleGrenoble / France


Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations

Scientists from the Institut de Biologie Structurale (IBS, a mixed research unit of the CEA, CNRS and Université Joseph Fourier), the ESRF, the University of Ulm (Germany) and the University of Southampton (United Kingdom) have just developed a new fluorescent protein derived from GFP (green fluorescent protein). The new protein, called Iris-FP, will help scientists to monitor the spatio-temporal dynamics of proteins using super-resolution optical microscopy. The results, recently published online by the journal Proceedings of the National Academy of Sciences raises exciting prospects for nanoscopy and biophotonics.

Nanoscopy is an emerging field in microscopy that allows samples to be imaged at spatial resolutions on the order of a few tens of nanometers, considerably higher than that possible by traditional optical microscopy.

One set of nanoscopic techniques makes use of fluorescent proteins that are derived from the natural protein GFP and have fluorescent properties which can be altered in a controlled manner.

Many structural biology groups are trying to improve these techniques by developing a new generation of fluorescent proteins. Some of these proteins have the property of being "photocommutable" : they can be switched on or off at will. Others are capable of photoconversion : their colour can be altered by exciting them with laser light.

The PNAS study reports the development of a new protein, Iris-FP, which combines both properties. Using ESRF X-rays, the research team determined the protein’s atomic structure and characterized each of its colour-states. Iris-FP is a highly versatile tool which promises to considerably advance microscopy techniques. By genetically fusing Iris-FP to a protein of interest, scientists will be able to monitor the protein’s movements within the cell at unprecedented spatial and temporal resolution.

Besides microscopy, the development of new fluorescent probes raises exciting prospects for nanotechnology. Potential future applications include the development of high-density mass storage media that exploit changes in the colour of crystals of these proteins, allowing a large amount of information to be stored in a nanometric-sized structure.

Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations. Virgile Adam, Mickaël Lelimousin, Susan Boehme, Guillaume Desfonds, Karin Nienhaus, Martin J. Field, Joerg Wiedenmann, Sean McSweeney, G. Ulrich Nienhaus, and Dominique Bourgeois. Proceedings of the National Academy of Sciences.), 105(47):18343-8

Young Researchers’ Award of the Foundation Bettencourt-Schueller awarded to Helena Paidassi

Helena Païdassi (former IBS PhD student) has been awarded one of the 14 prizes of the Foundation Bettencourt-Schueller. These prices of 21,000 euros are awarded annually to young researchers involved in life sciences for post-doctoral training courses abroad.

The bacterial cold-aminopeptidase and the human leukotriene A4 hydrolase, two close structural homologues

In this article the structure of an aminopeptidase from the psychrophilic bacterium Colwellia psychrerythraea (ColAP) was determined and compared with the "Leukotriene human A4 hydrolase" (LTA4H) structure, its nearest homologue. This structural analysis reveals two principal elements. From one hand, it highlights the various determinants implied in cold adaptation of the ColAP, in particular, an increase in the percentage of loops exposed to proteic surface and an increased flexibility of the C-terminal domain. From the other hand, the active sites comparaison of these enzymes suggests that the second activity of human enzyme would have appeared along evolution via a reshaping of the active site allowing it to convert the LTA4 into LTB4, a chemoattractant acting in inflammatory phenomena.

Crystal Structure of the Cold-active Aminopeptidase from the Arctic Bacterium Colwellia psychrerythraea, a close structural homologue of the human bifunctional leukotriene A4 hydrolase. Bauvois, C., Jacquamet, L., Huston, L.A., Borel, F., Feller, G., Ferrer, J.-L., JBC 283, 23315-23325.

2008 Science Fair : Open Days at the IBS and conference

Plaquette "Filles et garçons en sciences et techniques, un enjeu européen et planétaire"

The 17th edition of the Science Week tolok place in Isère from the 14th to the 23rd of November, 2008. For this edition the Institute organized two events :

- The Institute welcomed 180 secondary school pupils and invited them to discover the proteins which are essential for life. With the researchers, engineers and technicians of the IBS, they had the opportunity to visit laboratories, accomplish scientific experiments and discover the world of living on the atomic scale.

- Eva Pebay-Peyroula (director of the IBS) gave a talk during the conference entitled « Filles et garçons en sciences et techniques, un enjeu européen et planétaire ».

Photo credit : IBS/CEA-CNRS-UJF/O.Kaikati

Helical Structures of ESCRT-III Are Disassembled by VPS4

Eukaryotic cells maintain and adjust their cell surface protein composition in part by removing transmembrane proteins from the plasma membrane by endocytosis and delivering them to the lysosome for degradation. The proteins are sorted into vesicles, which bud off into the lumen of the endosome, giving rise to the multivesicular-body (MVB) compartment. The endosomal sorting system is conserved from yeast to mammals and includes ESCRT III (Endosomal Sorting Complexes Required for Transport) complexes. Part or all of the ESCRT machinery have been also implicated in other biological processes ranging from enveloped virus budding, cytokinesis, autophagy and neuron specific abnormalities. MVB vesicle sorting, enveloped virus budding and cytokinesis constitute topologically similar processes since they are budding processes directed away from the cytosol. All three processes require ESCRT-III complexes and the function of VPS4, a AAA-type ATPase. Thus a minimal common ESCRT-III function for all three processes is most likely membrane abscission to separate two membrane enveloped structures after completion of budding.

Our structural data (collaboration Pr Winfried Weissenhorn group, UVHCI and Dr Guy Schoehn UVHCI/IBS) show (by electron microscopy) that CHMP2A and CHMP3 assemble into large helical tubular structures that are disassembled by VPS4. The polymeric structures contain the VPS4 binding site on the inside and expose the membrane targeting surface on their outside, suggesting that the polymers assemble on the inside of a newly formed bud. We show further that CHMP2A and CHMP3 assemble on negatively charged phospholipids into a salt resistant complex. Such helical CHMP structures could thus assemble within the neck of an inwardly budding vesicle, catalyzing late steps in budding under the control of VPS4.

Helical structures of ESCRT-III are disassembled by VPS4.
Lata S, Schoehn G, Jain A, Pires R, Piehler J, Gottlinger HG and Weissenhorn W
Science (2008) 321(5894) : 1354-1357

CHMP 2A/3 complex
Left : Complex visualized by negative staining
Middle : Cryo EM of CHMP complex
Right : Low resolution 3D model based on cryoEM data
The scale bar represent 100 nm

The difference a Se makes ? Oxygen-Tolerant hydrogen production by the [NiFeSe]-hydrogenase from Desulfomicrobium baculatum.

Parkin, A., Goldet, G., Cavazza, C., Fontecilla-Camps, J.C. and Armstrong, F.A. (2008)

J Am Chem Soc. 130(40), 13410-6.

Kirstine Meyer prize awarded to Malene Ringkjøbing Jensen (FDP group)

Malene Ringkjøbing Jensen has been awarded the prestigious Kirstine Meyer prize for her research within protein chemistry and biology using nuclear magnetic resonance spectroscopy. She is currently a postdoc at the IBS in the group of Dr. Martin Blackledge (Protein Dynamics and Flexibility by NMR).

Kirstine Meyer (1861-1941) was Denmarks first female Ph.D in physics in 1909 (from the University of Copenhagen) . After her death, colleagues and friends established the prize in her memory. The Kirstine Meyer prize is awarded at least every second year to a young promising young Danish physicist or chemist.

Experimental approaches to kinetics of gas diffusion in hydrogenase

Fanny Leroux, Sebastien Dementin, Benedicte Burlat, Laurent Cournac, Anne Volbeda, Stephanie Champ, Lydie Martin, Bruno Guigliarelli, Patrick Bertrand, Juan Fontecilla-Camps, Marc Rousset, and Christophe Léger.

Proceedings of the National Academy of Sciences of the USA ,105(32):11188-93

Binding of ADP in the mitochondrial ADP/ATP carrier is driven by an electrostatic funnel

The ADP/ATP carrier (AAC) is a mitochondrial membrane protein, transporting ADP from the intermembrane space (IMS) to the matrix and ATP in the opposite direction. Based on the high-resolution structure of the bovine carrier, a total of 0.53 µs of classical molecular dynamics simulations were conducted.
The electrostatic funnel shown from three-dimensional mapping of the electrostatic potential forms a privileged passageway to drive ADP3- rapidly towards the bottom of the cavity. This was confirmed by several numerical experiments which revealed systematic association of ADP3- to the crevice of the AAC. Simulations relying on the use of an adaptive biasing force reveal for the ?rst time that the binding site corresponds to a free energy minimum in which the phosphate moiety disrupts one of three conserved salt bridges, probably the first event that triggers the conformational changes for the transport.

Binding of ADP in the mitochondrial ADP/ATP carrier is driven by an electrostatic funnel.
Dehez F, Pebay-Peyroula E and Chipot C
Journal of the American Chemical Society 130(38) : 12725-12733

Biomimetic Nanotechnology : A new type of biosensor

Researchers from the Institut de Biologie Structurale Jean-Pierre Ebel and the Institut de recherches en technologies et sciences pour le vivant , have introduced a new generation of biosensors. Using protein engineering, they have constructed proteins combining two properties : chemical signal recognition and its transduction into an electrical signal. These novel nanometric sensors could form the building blocks of miniature devices for drug screening, diagnostics, or toxic agent detection. This work was published in Nature Nanotechnology with a detailed editorial review [Abbas A & Roth BL (2008) Electrifying cell receptors. Nature Nanotech. 3:587-588].

- Press release.

- News & Views Nature Nanotech. 3:587-588

Coupling ion channels to receptors for biomolecule sensing.
Moreau CJ, Dupuis JP, Revilloud J, Arumugam K, Vivaudou M.
Nature Nanotech. 3:620-625

Principe de fonctionnement d'un ICCR (Ion Channel Coupled Receptor
The principle of ICCRs (Ion-Channel Coupled Receptors). In an ICCR, a natural receptor is attached to an ion channel so that a rigid mechanical link couples the 2 proteins. When the receptor recognizes a molecule X, it undergoes a structural change that is transmitted to the channel. The degree of opening of the channel is consequently modified and the ion flux through the channel is altered. This ion flux is easily detectable as an electrical current.

How intrinsically disordered proteins fold and bind : Quantitative NMR analyses of molecular recognition in Sendai virus Nucleoprotein

A significant fraction of proteins coded in the human proteome do not fold into stable three-dimensional structures but are either partially or completely unfolded. A key feature of this family of proteins is their proposed capacity to undergo a disorder-to-order transition upon interaction with a physiological partner. Although the observation of such events forces us to completely reassess our current understanding of protein-protein interactions in the cell, the mechanisms governing protein folding upon interaction, in particular the extent to which recognition elements are pre-configured prior to formation of molecular complexes, remains largely unknown.
NMR is the only technique that can probe the local behaviour of these highly flexible proteins, and the Blackledge group (Protein Dynamics and Flexibility by NMR) at the IBS has, over the last 5 years, developed novel techniques to characterize partially folded proteins in terms of rapidly interchanging conformational ensembles.
In collaboration with the Ruigrok group at the UVHCI, the most recent developments have been applied to generate a quantitative description of the partially folded C-terminal domain of Sendai virus nucleoprotein (NTAIL) in solution. Rather than fraying randomly, the molecular recognition element of NTAIL preferentially populates three specific overlapping helical conformers, each stabilized by an N-capping interaction prior to interaction. This study provides experimental evidence for the molecular basis of helix formation in partially folded peptide-chains, carrying clear implications for both molecular recognition and the early steps of protein folding.

On the Origin of NMR Dipolar Waves in Transient Helical Elements of Partially Folded Proteins. Jensen, M. R. ; Blackledge, M. J. Am. Chem. Soc. ; 2008 ; 130 ; 11266-11267.

Quantitative Conformational Analysis of Partially Folded Proteins from Residual Dipolar Couplings : Application to the Molecular Recognition Element of Sendai Virus Nucleoprotein. Jensen, M. R. ; Houben, K. ; Lescop, E. ; Blanchard, L. ; Ruigrok, R. W. H. ; Blackledge, M. J. Am. Chem. Soc. ; 2008 ; 130 ; 8055-8061.

Anti-parallel Microtubule bundling promoted by two Arabidopsis Thaliana proteins

In plant cells, interphase microtubules occur predominantly at the cell cortex as ordered bundles in close association with the plasma membrane. These microtubules play crucial roles in cell morphogenesis and are usually transversely oriented relative to the longest cell axis. At the molecular level, putative candidate proteins stimulating the cross-linking of MTs in vitro and in vivo have been identified. They are mainly members of the MT-associated family MAP65s (« Micotubule-Associated Protein 65 »). In Arabidopsis we showed that two AtMAP65s members associated with MTs and promoted the bundling of anti-parallel aligned MTs in vitro.

Two Microtubule-Associated Proteins of Arabidopsis MAP65s promote anti-parallel microtubule bundling. Gaillard J*,Neumann E*, Van Damme D, Stoppin-Mellet V, Ebel C, Barbier E, Geelen D, Vantard M. * both authors contributed equally to this work.Mol Biol Cell. 2008, Jul 30.

Observing the target enzyme of Alzheimer drugs at work

Observing the enzyme acteylcholinesterase at work to understand its molecular mode of action – this is the objective of scientists at the Institut de Biologie Structurale in Grenoble who published their results in the Proceedings of the National Academy of Science of the USA. In collaboration with their colleagues from the ESRF, the IBPC in Toulouse and the Weizmann Institute of Science in Israel, they observed structural changes in one of Nature’s fastest enzymes during its catalytic cycle. Understanding the structural dynamics of acetylcholinesterase, a major target for anti-Alzheimer medication, is an important prerequisite for the design of more efficient drugs.

Shoot-and-trap : use of specific X-ray damage to study structural protein dynamics by temperature-controlled cryo-crystallography.
Colletier, J.-P., Bourgeois, D., Sanson, B., Fournier, D., Silman, I., Sussman, J.L. & Weik, M.
Proc. Natl. Acad. Sci. 2008 Aug 19 ;105(33):11742-11747.

Figure legend : After cleavage of an analogue of the neurotransmitter acetylcholine (yellow sticks) in the active site of acetylcholinesterase (blue sticks), choline, i.e. one of the cleavage products, reorients in the enzymatic active site. The reorientation of choline, from the red to the green position, is sketched by the black arrow

An agreement between the IBS and the Forschungszentrum Jülich was signed on July 3, 2008

Following the contract signed in February 2008 between the CEA and the Helmholtz Gemeinschaft (Association of German Research Centres), the IBS has signed a specific agreement for cooperation with the Forschungszentrum Jülich GmbH (FZJ).

This agreement is expected to lead to strong interactions in the areas of membrane proteins and proteins involved in neurodegenerative diseases

Dr. Pierre Legrain (director of the Direction des Sciences du Vivant of the CEA) and Dr. Sebastian Schmidt (member of the Board of Directors of the FZJ responsible for Key Technologies and the Structure of Matter)

Prof. Eva Pebay-Peyroula (director of the IBS) and Prof. Dieter Willbold (director of the Institute of Molecular Biophysics at the FZJ)

A project from the IBS awarded in a competition supporting the creation of startup companies with innovative technological products

For 10 years, a French national competition has existed that identifies and helps finance the creation of startup companies with innovative technological products.

The award ceremony this year was held in Lyon on the 19th June 2008 and presided over by the Minister of Higher Education and Research, Madame Valérie Pécresse. Of the housand or so entries that were in competition, 170 received a prize. Among these was the NatX project from the IBS which was one of the winners in the "emerging technologies" category.

This project, piloted by Nathalie Ferrer, and aided by Jean-Luc Ferrer, Xavier Vernede and Franck Borel, aims to commercialize automated systems, and associated services, 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.

Structure Elucidation of a Kissing Complex and Application to the AIDS Virus

Scientists at the Jean-Pierre Ebel Institute of Structural Biology in collaboration with teams at the European Institute of chemistry and biology and Ottawa University have used innovating NMR techniques to elucidate the specific recognition mechanisms between AIDS virus RNA and a synthetic RNA. These results, published on July 8th in the review Proceedings of the National Academy of Sciences of USA, should provide a basis for the development of new therapies targeting viral RNA sequences...[Press release]

Liquid Crystal NMR Structure of HIV TAR RNA Bound to its SELEX RNA Aptamer Reveals the Origins of the High Stability of the Complex.
Van Melckebeke, V., Devany, M., Di Primo, C., Beaurain, F., Toulmé, J.J., Bryce, D. & Boisbouvier J.
Proc. Natl. Acad. Sci. USA, 105, 9210-9215 (2008).

Structural Basis for the Stability of the HIV TAR-aptamer Kissing Complex Revealed

CNRS 2008 Bronze Medal awarded to Jerome Boisbouvier (LRMN)

Jerome Boisbouvier (LRMN) is the recipient of one of the CNRS bronze medals for the year 2008. This distinction is meant to reward an ongoing and fruitful research activity and represents an encouragement from the CNRS to pursue it.

About 50 bronze medals are awarded annually (list of winners 2008).

Third IBS Science day

June 27, amphitheatre "Ouest", UFR de Chimie, on the University Campus

This event was open to all members of the IBS staff. The objective of this manifestation was to facilitate the discovery of the activities carried out in the context of the major research topics of the IBS, and to present new technologies available at the Institute. In addition to conferences, second- and third-year PhD students were presenting their work in the form of posters. A prize for the best poster was awarded.

Conference in the seminar room

Posters sessions

Protein crystallography through supramolecular interactions between a lanthanide complex and arginine

Pompidor G, D’Aleo A, Vicat J, Toupet L, Giraud N, Kahn R and Maury O.

Angew Chem Int Edit, 47 : 3388-3391

Structure of tumor suppressor p53 and its intrinsically disordered N-terminal transactivation domain

Wells M, Tidow H, Rutherford TJ, Markwick P, Jensen MR, Mylonas E, Svergun DI, Blackledge M and Fersht AR.

P Natl Acad Sci USA, 105 : 5762-5767.

Toward the characterization of peptidoglycan structure and protein-peptidoglycan interactions by solid-state NMR spectroscopy

Kern T, Hediger S, Muller P, Giustini C, Joris B, Bougault C, Vollmer W and Simorre JP.

Journal of the American Chemical Society, 130 : 5618-5619

Stabilization by extra-helical thymines of a DNA duplex with Hoogsteen base pairs

Joan Pous, Lourdes Urpí, Juan A. Subirana, Catherine Gouyette, Jorge Navaza and J.Lourdes Campos.

Journal of the American Chemical Society, 130(21):6755-60.

Hadamard amino-acid-type edited NMR experiment for fast protein resonance assignment.

Lescop E, Rasia R and Brutscher B.

Journal of the American Chemical Society, 130 : 5014-5015

How plants avoid shade

Rapid Synthesis of Auxin via a New Tryptophan-Dependent Pathway Is Required for Shade Avoidance in Plants.

Yi Tao, Jean-Luc Ferrer, Karin Ljung, Florence Pojer, Fangxin Hong, Jeff A. Long, Lin Li, Javier E. Moreno, Marianne E. Bowman, Lauren J. Ivans, Youfa Cheng, Jason Lim, Yunde Zhao, Carlos L. Ballare´ , Go¨ ran Sandberg, Joseph P. Noel, and Joanne Chory.

Cell (2008), 133 : 164–176.

[See press release]

Motions of soluble protein are slaved to hydration-water dynamics

The thin film of water around proteins, their hydration water, is vital to the macromolecule’s biological activity. Without hydration water, proteins lack the conformational flexibility that animates their 3D structures and brings them to life. Consequently, protein dynamics is thought to be slaved to hydration-water dynamics. There is growing experimental and computational evidence that protein and water dynamics are indeed intimately coupled. One way of exploring the coupling exploits so-called dynamical transitions, characteristic changes in atomic fluctuations that appear in both water and proteins if studied as a function of temperature. Do both dynamical transition temperatures coincide ? In other words, does a transition in the water trigger one in the protein at the same temperature ? If the coupling is indeed tight, one would assume so, but direct experimental proof has been missing.

Scientists at the Institut de Biologie Structurale and the ILL, in collaboration with the University of California and the University of Perugia, combined incoherent neutron scattering experiments and MD simulations on a soluble protein to gain insight into the dynamical coupling between protein and hydration-water dynamical transitions. The experimental trick played to directly access the water transition was perdeuteration of the protein at the ILL-EMBL Deuteration Laboratory, which almost completely masked the protein’s contribution to the neutron scattering signal. A hydration-water transition was observed at 220 K, coinciding with the dynamical transition of the protein. The latter was measured on a hydrogenated protein sample hydrated in D2O. MD simulations reproduced the coincidence of transitions and revealed that both originate from the onset of translational diffusion of water molecules at the protein’s surface. The unprecedented combination of neutron scattering, perdeuteration and MD simulations, carried out on the same protein, paints a coherent picture in which the dynamics of soluble proteins and their hydration-water are strongly coupled. This strikingly contrasts the case of membrane proteins, the dynamics of which is controlled by lipids rather than by water dynamics (Wood et al. (2007) PNAS 104, 18049).

Soluble protein in red and hydration water in blue. The graph shows amplitudes of thermal motions as a function of temperature. Hydration-water dynamics and soluble-protein dynamics both have a similar temperature dependence. Unlike membrane proteins, soluble proteins are dynamically dominated by hydration water.

Coincidence of dynamical transitions in a soluble protein and its hydration water : direct measurements by neutron scattering and MD simulations.
Wood, K., Frölich, A., Paciaroni, A., Moulin, M., Härtlein, M., Zaccai, G., Tobias, D. & Weik, M. (2008).
J. Am. Chem. Soc. 2008 Mar 14 [Epub ahead of print]

Penicillin-binding proteins and beta-lactam resistance

The shape of bacteria is determined by that of their cell wall. Streptococci, lactococci and enterococci are the same shape and share the same set of penicillin-binding proteins, the enzymes that assemble the peptidoglycan, the main constituent of the cell wall. Assuming that the common morphology of these organisms arises from a common mechanism, the present review aims to integrate older ultrastructural literature, pertaining mostly to enterococci, and recent localization studies in pneumococci. Two machineries of peptidoglycan synthesis are proposed to function at the septum.

Penicillin-binding proteins and beta-lactam resistance.
Zapun A, Contreras-Martel C and Vernet T
FEMS Microbiol Rev. 2008 Mar ;32(2):361-85

The different shapes of cocci

The shape of bacteria is determined by their cell-wall and can be very diverse. Even among genera with the suffix “cocci” different shapes exist. While staphylococci or neisseria cells, for example, are truly round-shapped, streptococci, lactococci or enterococci have an ovoid shape. Interestingly, there seems to be a correlation between the shape of an organism and its set of penicillin-binding proteins - the enzymes that assemble the peptidoglycan, the main constituent of the cell-wall. While only one peptidoglycan biosynthesis machinery seems to exist in staphylococci, two of these machineries are proposed to function in ovoid-shaped bacteria, reinforcing the intrinsic differences regarding the morphogenesis of different classes of cocci. The integration of older ultrastructural literature and recent localization studiesclarifies the relation between the mechanisms of cell-wall synthesis and the determination of cell shape in various cocci.
The different shapes of cocci.
Zapun A, Vernet T and Pinho MG.
FEMS Microbiol Rev. 2008 Mar ;32(2):345-60

C1q binds phosphatidylserine and likely acts as a multiligand-bridging molecule in apoptotic cell recognition

Païdassi H, Tacnet-Delorme P, Garlatti V, Darnault C, Ghebrehiwet B, Gaboriaud C, Arlaud GJ, Frachet P.
Journal of Immunology, 180 : 2329-2338

Press briefing for the official presentation of an ARC grant to an IBS team

A press briefing was held at the IBS on Monday, Feb. 11 for the official presentation of a grant from the Association pour la Recherche sur le Cancer (ARC) awarded to a research project on a protein involved in pancreatic cancer.

The project is entitled "Etude fonctionnelle et structurale des récepteurs du leucotriène B4 pris comme nouvelles cibles thérapeutiques dans le cancer du pancréas". It involves three research groups :
- Laurence SERRE (Institut de Biologie Structurale – Grenoble),
- Bernard PUCCI (Laboratoire de Chimie Bio-Organique – Avignon),
- Jean-Louis BANERES (Faculté de Pharmacie – Montpellier).

The award presentation took place in the presence of Jacques Raynaud, Chairman of the ARC, the researchers Laurence SERRE, Bernard PUCCI and Jean-Louis BANERES, journalists and institutional representatives.

Crédit photo : IBS/CEA-CNRS-UJF