2013

How do fluorescent proteins die ?

Fluorescent proteins are widespread markers in cellular imaging, providing a highly flexible toolbox to investigate live cells. Unfortunately, contrary to organic dyes, fluorescent proteins are particularly sensitive to the photobleaching phenomenon, the definitive loss of fluorescence following photo-induced destruction of the chromophore. Photobleaching is particularly problematic in super-resolution microscopy techniques, which are being rapidly developed today, limiting the resolution that can be achieved. By combining kinetic crystallography, optical and Raman spectroscopy, molecular dynamics simulations, mass spectrometry, and super resolution microscopy, we have investigated the photophysical mechanisms leading to photobleaching of the fluorescent protein IrisFP. We have shown that depending on the illumination intensity used for the imaging experiment, two completely different photobleaching mechanisms show up. At low laser intensity, typical of a standard widefield microscopy experiment, an oxygen-dependent mechanism predominates. On the contrary, at high laser intensity, typical of super-resolution microscopy experiments, a redox-dependent mechanism prevails. The first mechanism, which generates reactive oxygen species (ROS) in the cell is thus expected to be more cytotoxic than the second mechanism, which does not generate such species. Thus, this work suggests in a counterintuitive manner that by increasing laser intensity at constant those, less cellular damages would be created. This hypothesis now needs to be experimentally verified.

Structural evidence for a two-regime photobleaching mechanism in a reversibly switchable fluorescent protein.
Duan C, Adam V, Byrdin M, Ridard J, Kieffer-Jaquinod S, Morlot C, Arcizet D, Demachy I, Bourgeois D.
J Am Chem Soc. 2013 Oct 23 ;135(42):15841-50. doi : 10.1021/ja406860e. Epub 2013 Oct 7.

Choreographed origami : folding ribosomal RNA involves paired tagging sequence

Building ribosomes – the cell’s protein factories – is like a strictly choreographed dance. Other ‘machines’ inside the cell have to produce specific RNA molecules and fold them into the right shape, then combine the folded RNA with proteins to form a working ribosome. The study combined nuclear magnetic resonance experiments performed at EMBL and neutron scattering experiments performed at the ILL in Grenoble, France.
To know more

The structure of the box C/D enzyme reveals regulation of RNA methylation.
Lapinaite A, Simon B, Skjaerven L, Rakwalska-Bange M, Gabel F, Carlomagno T.
Nature. 2013 Oct 24 ;502(7472):519-23. doi : 10.1038/nature12581. Epub 2013 Oct 13.

Modelling of chemical reactions in the spotligh

Chemists used to create models of molecules using plastic balls and sticks. Today the modelling is carried out in computers, thanks in large part to the work done in the 1970s by Martin Karplus, Michael Levitt and Arieh Warshel. They developed powerful computer models that are used all around the world to understand and predict chemical processes. Applications of their discoveries are endless for both research and industry. " Detailed knowledge about chemical processes permits the optimization of catalysts for cars or the design of drugs and materials for solar cells" the Swedish Royal Academy of Sciences said.

The theoretical work of the Nobel Laureates focused on molecular dynamics in general, and hybrid methods in particular. Martin Field (IBS/DYNAMOP), a postdoctorate in the laboratory of Martin Karplus, played a very important role in the initial development of hybrid methods (method coupling classical and quantum mechanics). Today his DYNAMO team and Patricia Amara (IBS/METALLO) continue this work in the IBS to study the structure and function of proteins and other biological macromolecules and their complexes.

UJF prize awarded to F.X. Gallat

François-Xavier Gallet won the Biology-Chemistry interface PhD thesis prize of the Chemistry and Biology Faculty at the Université Joseph Fourier (UJF). This prize honours PhD students who published their work as first authors in high-profile journals. During his PhD thesis, François-Xavier studied the dynamics of intrinsically disordered proteins and of protein-polymer nanohybrids by neutron scattering and complementary methods.

Move to EPN Campus underway : exciting time ahead

Crédit photo : CEA/D. Morel

From its initial size of 100 people, within 20 years the IBS has grown to an institute of over 240 individuals and with its installation on EPN campus (European Photon and Neutron science campus), continues on an international track.

The new building of 9500 m2 is now ready to host new teams or start-up companies and will provide closer proximity to the European PSB partners (Partnership for Structural Biology). For one month the IBS will be a hive of activity with the move of researchers and platforms.
Cutting-edge platforms developed within FRISBI (Investissements d’Avenir grant) in the context of Instruct (European network of Integrated Structural Biology) will thus be on a single site and will offer access to high-field NMR spectrometers, electron microscopes, and several platforms for sample production and characterization, that are complementary to X-rays and neutron sources provided by ESRF and ILL.

Please note our new address :
6 rue Jules Horowitz, 38000 Grenoble - 04 57 42 85 00
Site access : Visitors to the EPN campus must be registered for a visit prior to arrival and need to produce proof of identification.

The NMR-Bio development project awarded by ERC

Teams of Jerome Boisbouvier (IBS/NMR) and Olivier Hamelin (iRTSV) jointly develop technology solutions in isotopic labelling to push forward Biomolecular NMR frontiers. With support from CNRS, CEA and Grenoble Alps Innovation, the consortium had transformed these basic research findings into innovative market products that are now distributed by the CEA (www.nmr-bio.com). The project has been selected by the proof of concept program of the European Research Council in order to set up an independent company valuing these Grenoble inventions (patents from CEA / CNRS / UJF) into products and innovative services.

NMR-Bio is the third project from IBS Biomolecular NMR spectroscopy group selected by ERC (after SeeNanoLifeInAction ERC-consolidator grant awarded to J. Boisbouvier in 2010 and ProtDyn2Function ERC-starting grant awarded to Paul Schanda in 2012).

Contact IBS researcher : : Jérome Boisbouvier

Creation of an International Associated Laboratory between France (Grenoble) and Brazil (Campinas)

An agreement creating an International Associated Laboratory (LIA) involving Ibs (for the French side) and LNBio and CNPEM (for the Brazilian side) was signed. This intenational associated laboratory named "BACWALL" aims to study the assembly and structure of macromolecular complexes involved in the synthesis of bacterial cell wall and virulence. This work may lead to significant advances in the understanding of bacterial virulence and thus develop new antibiotics.

Coping mechanisms of pneumococcus

Bacterial cell division requires the co-ordinated action of machineries composed by peptidoglycan biosynthetic enzymes and cell morphogenesis proteins. The regulation of these processes, notably in ovococci remains largely uncharacterized. The conserved eukaryotic-like Ser/Thr protein kinase of Streptococcus pneumoniae (StkP) plays a major role in cell shape and division. The molecular mechanisms underlining the regulatory function(s) of StkP through its interaction with the essential actor of septal peptidoglycan synthesis Penicillin-Binding Protein 2x (PBP2x) was investigated. We show that StkP and PBP2x interact directly in vitro and are present in the same membrane-associated complex in S. pneumoniae. We further show that they both display a late-division localization pattern at the division site and that their positioning is independent from each other. We demonstrate that StkP and PBP2x interaction is mediated by their extracellular regions and that the complex is dissociated in presence of muropeptides. All together, these data suggest a model in which StkP regulates cell division in the pneumococcus through the control of the septal peptidoglycan synthesis machinery.

Interaction of Penicillin-Binding Protein 2x and Ser/Thr protein kinase StkP, two key players in Streptococcus pneumoniae R6 morphogenesis. Morlot C, Bayle L, Jacq M, Fleurie A, Tourcier G, Galisson F, Vernet T, Grangeasse C, Di Guilmi AM. Molecular Microbiology, 2013 Oct ;90(1):88-102

The 2013 Walter Hälg Prize awarded to Joe Zaccai

Giuseppe Zaccai (IBS) has just been awarded the prestigious Walter Hälg prize for an outstanding, coherent work in neutron scattering with long-term impact on scientific and/or technical neutron scattering applications.

The European Neutron Scattering Association underlined his “pioneering contributions to the application of neutron scattering to a range of biophysical and biochemical problems in biology, which has provided important insights in the debate on the relationship between molecular structure and dynamics and biological function, and for his leading advocacy of the role of neutron scattering in biological research.”

10th anniversary celebrations for Grenoble’s Partnership for Structural Biology (PSB)

Structural biology is one of the central tenets of research in Grenoble along with micro/nano technologies and energy. In 2003, three European and two French institutes joined forces to create the PSB and pool their knowledge and state-of-the-art equipment to study structural biology.
The PSB has accompanied the development and explosion of this discipline in Grenoble. Structural biology examines how proteins and nucleic acids acquire their shape, and how alterations may affect their function. A deeper understanding of molecules and their properties is essential, for example, for the development of new and efficient medicine.
The PSB provides a unique palette of tools for structural biology covered by 14 technical platforms spanning sample expression and crystallisation, as well as structure resolution, and imaging.
In the past 10 years, the PSB has been the scene of several major discoveries :

• The development of kinetic crystallography, a technique that shows how proteins function
• A mechanism that enables viral budding at the surface of infected cells
• The discovery of the three-dimensional structure of the flu virus polymerase. Knowledge of this structure opens new routes in the discovery and development of anti-flu drugs

Photos of the event

Members of the PSB :

• ESRF – European Synchrotron Radiation Facility
• EMBL – the Grenoble outstation of the European Molecular Biology Laboratory
• ILL – Institut Laue Langevin
• IBS – Institut de Biologie Structurale
• UVHCI – Unit of Virus Host Cell Interactions (UJF-EMBL-CNRS)

Recent insights into the function of human complement component C1q

C1q is a recognition protein able to sense a wide variety of immune and non immune targets, including pathogens and damaged structures from self, and to trigger the classical complement pathway through activation of its associated proteases C1r and C1s. Precise mapping of the ligand binding sites of C1q has been precluded so far due to the unavailability of recombinant C1q, a complex protein assembled from 18 polypeptide chains of three different types encoded by three genes.
We have successfully expressed full-length recombinant C1q in mammalian cells. The resulting rC1q molecule is similar to serum C1q as judged from biochemical and functional analyses and exhibits the characteristic shape of a bunch of flowers. Using site-directed mutagenesis, we have identified two lysine residues located in the B and C chains that play a key role in the interaction with C1r and C1s. The availability of rC1q opens the way for deciphering the molecular basis of the unusual binding versatility of this protein by mapping the residues involved in the sensing of its multiple targets and the binding of its receptors.

Expression of recombinant human complement C1q allows identification of the C1r/C1s-binding sites, Bally I, Ancelet S, Moriscot C, Gonnet F, Mantovani A, Daniel R, Schoehn G, Arlaud GJ, Thielens NM. Proc Natl Acad Sci U S A, 2013 May 21 ;110(21):8650-5

Looking at bacterial cell walls with NMR

The cell wall of bacteria is composed of several biopolymers building a shell around the cell, which allows recognition and adhesion to hosts, as well as regulation of other important cellular functions. A better understanding of the complex cell wall structure and interactions may help scientist to develop new antibiotics. Solid-state nuclear magnetic resonance (NMR) offers an interesting tool to look at this huge and complex molecular system in intact conditions, as it is principally not limited by the size of the object and do not require any crystallization. However, it suffers from its intrinsic low sensitivity, especially for the case of cell wall studies in living cells. Using high-field dynamic nuclear polarization (DNP), an emerging technique aimed at improving sensitivity of NMR, researchers of INAC and IBS demonstrate that signal intensity from the bacterial cell wall can be 24-fold enhanced, opening the possibility of atomic-scale studies of cell-wall interactions.
To obtain this gain in sensitivity, the research teams of INAC and IBS mixed Bacillus subtilis bacterial cells with a biradical commonly used for DNP and called TOTAPOL, which has the ability to hyperpolarize nearby nuclei when properly irradiated with micowaves, boosting thus the NMR signal. This interdisciplinary team demonstrated that TOTAPOL molecules stick selectively to the cell-wall polymers. As the radical only polarizes nuclei in a certain proximity, this binding affinity allows the researchers to enhance selectively signals from the cell wall in living cells, or in the opposite to suppress them by saturation, leaving only signals from inside the cell. In addition to this selectivity, they discover that the combination of spectra acquired at different radical concentrations increases the spectral resolution, one of the bottlenecks of DNP-enhanced solid-state NMR, by removing some broad signal components. This study opens new opportunity to investigate more generally cell surfaces or other compartments in living cells by adjusting the radical affinity to the polymers of interest.

Solid-State NMR on Bacterial Cells : Selective Cell Wall Signal Enhancement and Resolution Improvement using Dynamic Nuclear Polarization. Hiroki Takahashi, Isabel Ayala, Michel Bardet, Gaël De Paëpe, Jean-Pierre Simorre, and Sabine Hediger. J Am Chem Soc. 2013 Feb 12

Cryo-electron microscopy three-dimensional structure of a phage infecting a bacteia

ϕRSL1 jumbo phage belongs to a new class of viruses within the Myoviridae family. Here, we report its three-dimensional structure determined by electron cryo microscopy.

Cryo-electron microscopy three-dimensional structure of the jumbo phage ΦRSL1 infecting the phytopathogen Ralstonia solanacearum. Effantin G, Hamasaki R, Kawasaki T, Bacia M, Moriscot C, Weissenhorn W, Yamada T, Schoehn G. Structure, 2013 Feb 5 ;21(2):298-305

Features responsible for O2 tolerance of membrane-bound NiFe-hydrogenases

Most NiFe-hydrogenases are sensitive to oxygen (O₂) damage of their bimetallic active site. However, there is a subclass that, being exceptionally tolerant to O₂, can oxidize hydrogen (H₂) under air. The IBS/Metalloproteins Unit, in collaboration with Prof. Armstrong’s group at Oxford University, has solved the crystal structure of hydrogenase 1 (Hyd-1) from Escherichia coli in complex with its physiological partner cytochrome b (1). Hyd-1 exhibits several features to tolerate O₂ : i) an unprecedented [4Fe3S]6Cys cluster with a distorted geometry ; calculations, performed in collaboration with Dr. Mouesca (CEA/INAC), show that this plasticity explains how a superoxidation is achieved at a surprisingly low redox potential (2) ; ii) the dimeric hydrogenase minimizes O₂-induced active site inactivation through a repair mechanism where electrons produced at the functioning active site in one monomer flow to its O₂-exposed counterpart in the other and reduce O2 to water (1). These studies should help designing H₂ catalysts with improved performances under oxic conditions.

1. Crystal structure of the O₂-tolerant membrane-bound hydrogenase 1 from Escherichia coli in complex with its cognate cytochrome b. Volbeda A, Darnault C, Parkin A, Sargent F, Armstrong FA and Fontecilla-Camps JC, Structure, 21 : 184-190 (2013).
2. The structural plasticity of the proximal [4Fe3S] cluster is responsible of O₂ tolerance of membrane-bound [NiFe] hydrogenases. Mouesca JM, Fontecilla-Camps JC and Amara P, Angew. Chem. Int. Ed., DOI : 10.1002/anie.201209063.

Creation of the “Integrated Structural Biology, Grenoble” mixed research institute

Development of integrated structural biology at the Grenoble and European scale
The new challenge in structural biology is to understand biological processes at the molecular level in a cellular context (or even broader). To accomplish this, integrated structural biology is based on a collection of high-tech experimental approaches, and requires equipment (synchrotron radiation, high-field NMR, cryo-electron tomography) that is sometimes expensive. This also involves multidisciplinary expertise (biology, physics, chemistry) ranging from the production of samples to their characterization (structure, assembly and architecture, dynamics, function, interactions, etc.). Thanks to the presence of large equipment, Grenoble pioneered these approaches and established the PSB (Partnership for Structural Biology) as early as 2002, linking the ESRF, ILL, EMBL and IBS research teams ; more recently, they have been joined by teams from the UVHCI [1]. This first step has made possible the pooling of resources, and new concerted developments.

In general, it is now accepted that this type of biology requires a collection of facilities corresponding to the equivalent of large instruments. The aim of the European initiative ESFRI is to establish such infrastructures. Within this context, the project Instruct (Integrating Structural Biology), which began in 2011, defines 15 reference centers for integrated structural biology in Europe and 5 affiliated centers. Grenoble, and more specifically the combination of the two French units of the PSB (IBS and UVHCI), represents one of the major reference centers for Instruct.

The project FRISBI (French Infrastructure for Integrated Structural Biology), comprised of the two French Instruct centers (Grenoble and Strasbourg) as well as three other nationally important structural biology centers, was selected as part of the program “Investissement d’Avenir”. Grenoble has thus received 11.2 million € to support major improvements in most of the PSB facilities, notably including the purchase of new NMR spectrometers and electron microscopes.

The missions of the new UMS
PSB’s mode of concerted decision making, sufficient for a local pooling of facilities, needed to be formalized to meet the needs of the scientific community. The boundaries of the facilities, associated personnel, modes of operation and types of services for each facility, as well as access conditions (selection of projects, costs, management, etc.) have been specified for the different users. All technological facilities from the IBS thus obtained in July 2011 the ISO 9001 certification for their activities in production and purification of proteins, the characterization of their structure and biophysical properties, dynamics and assembly by X-ray, NMR and microscopy.

Darren Hart (EMBL) has been appointed director of the new Unité Mixte de Service, and is assisted by Yvette Gaude in financial administration. The governance of the UMS will involve the management of IBS and UVHCI within a local steering committee. The vast majority of scientific and technical personnel working at the facilities will keep their affiliation with the IBS and UVHCI research teams, to allow the best possible interaction between facility and research. The equipment present on the day of the creation of the UMS will thus evolve according to the needs of scientific projects and the UMS will ensure the sustained operation of the facilities by guaranteeing optimum access to all types of users.