Etude biochimique et structurale du complexe b6f, du complexe membranaire de translocation de la voie Sec et du complexe FhuA-pb5.

A mon arrivée en septembre 1993 au laboratoire de Photosynthèse de l’Institut de Biologie Physico-Chimique dans l’équipe de Jean-Luc Popot, je me suis intéressée à la biochimie des protéines membranaires : i) le mécanisme d’inactivation par les détergents, et ii) la caractérisation en terme de sous-unités (nombre, topologie), cofacteurs, état d’oligomérisation d’un complexe purifié, le b 6 f de la chaîne photosynthétique. Ceci m’a mené à en étudier la structure à partir de cristaux bidimensionnels lors d’un stage post-doctoral à Francfort dans le labo de Werner Kühlbrandt, mais également, à mon retour en France, à développer de nouveaux tensioactifs (hémi)fluorés pour la biochimie des protéines membranaires. De mon séjour en Allemagne j’ai rapporté le goût et la culture de la structure des protéines membranaires, avec la résolution de la structure à 8Å du complexe SecYEG, le translocon de la membrane interne de E. coli. Je m’intéresse donc maintenant également au mécanisme de reconnaissance phage-bactérie et de l’injection de l’ADN phagique dans la bactérie d’un point de vue structural

New insights into pb5, the receptor binding protein of bacteriophage T5, and its interaction with its Escherichia coli receptor FhuA

International audienceThe majority of bacterial viruses are bacteriophages bearing a tail that serves to recognise the bacterial surface and deliver the genome into the host cell. Infection is initiated by the irreversible interaction between the viral receptor binding protein (RBP) and a receptor at the surface of the bacterium. This interaction results ultimately in the phage DNA release in the host cytoplasm. Phage T5 infects Escher-ichia coli after binding of its RBP pb5 to the outer membrane ferrichrome transporter FhuA. Here, we have studied the complex formed by pb5 and FhuA by a variety of biophysical and biochemical techniques. We show that unlike RBPs of known structures, pb5 probably folds as a unique domain fulfilling both functions of binding to the host receptor and interaction with the rest of the phage. Pb5 likely binds to the domain occluding the b-barrel of FhuA as well as to external loops of the barrel. Furthermore, upon binding to FhuA, pb5 undergoes conformational changes, at the secondary and tertiary structure level that would be the key to the transmission of the signal through the tail to the capsid, triggering DNA release. This is the first structural information regarding the binding of a RBP to a proteic receptor. Ó 2012 Elsevier Masson SAS. All rights reserved

Assessing the conformational changes of pb5, the receptor-binding protein of phage T5, upon binding to its Escherichia coli receptor FhuA

Within tailed bacteriophages, interaction of the receptor-binding protein (RBP) with the target cell triggers viral DNA ejection into the host cytoplasm. In the case of phage T5, the RBP pb5 and the receptor FhuA, an outer membrane protein of Escherichia coli, have been identified. Here, we use small angle neutron scattering and electron microscopy to investigate the FhuA-pb5 complex. Specific deuteration of one of the partners allows the complete masking in small angle neutron scattering of the surfactant and unlabeled proteins when the complex is solubilized in the fluorinated surfactant F6-DigluM. Thus, individual structures within a membrane protein complex can be described. The solution structure of FhuA agrees with its crystal structure; that of pb5 shows an elongated shape. Neither displays significant conformational changes upon interaction. The mechanism of signal transduction within phage T5 thus appears different from that of phages binding cell wall saccharides, for which structural information is available

Plastid thylakoid architecture optimizes photosynthesis in diatoms

Photosynthesis is a unique process that allows independent colonization of the land by plants and of the oceans by phytoplankton. Although the photosynthesis process is well understood in plants, we are still unlocking the mechanisms evolved by phytoplankton to achieve extremely efficient photosynthesis. Here, we combine biochemical, structural and in vivo physiological studies to unravel the structure of the plastid in diatoms, prominent marine eukaryotes. Biochemical and immunolocalization analyses reveal segregation of photosynthetic complexes in the loosely stacked thylakoid membranes typical of diatoms. Separation of photosystems within subdomains minimizes their physical contacts, as required for improved light utilization. Chloroplast 3D reconstruction and in vivo spectroscopy show that these subdomains are interconnected, ensuring fast equilibration of electron carriers for efficient optimum photosynthesis. Thus, diatoms and plants have converged towards a similar functional distribution of the photosystems although via different thylakoid architectures, which likely evolved independently in the land and the ocean.ISSN:2041-172

Target highlights in CASP14 : Analysis of models by structure providers

Abstract The biological and functional significance of selected CASP14 targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modelled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins. This article is protected by copyright. All rights reserved.Peer reviewe

Protein target highlights in CASP15: Analysis of models by structure providers

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology

Conformational changes in the cytochrome b6f complex induced by inhibitor binding.

International audienceBinding of stigmatellin, an inhibitor of the Q(o) site of the bc-type complexes, has been shown to induce large conformational changes of the Rieske protein in the respiratory bc(1) complex (Kim, H., Xia, D., Yu, C. A., Xia, J. Z., Kachurin, A. M., Zhang, L., Yu, L., and Deisenhofer, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8026-8033; Iwata, S., Lee, J. W., Okada, K., Lee, J. K., Iwata, M., Rasmussen, B., Link, T. A., Ramaswamy, S., and Jap, B. K. (1998) Science 281, 64-71; Zhang, Z., Huang, L., Shulmeister, V. M., Chi, Y. I., Kim, K. K., Hung, L. W., Crofts, A. R., Berry, E. A., and Kim, S. H. (1998) Nature 392, 677-684). Such a movement seems necessary to shuttle electrons from the membrane-soluble quinol to the extramembrane heme of cytochrome c(1). To see whether similar changes occur in the related photosynthetic b(6)f complex, we have studied the effect of the binding of stigmatellin to the eukaryotic b(6)f complex by electron crystallography. Comparison of projection maps of thin three-dimensional crystals prepared with or without stigmatellin, and either negatively stained or embedded in glucose, reveals a similar type of movement to that observed in the bc(1) complex and suggests also the occurrence of conformational changes in the transmembrane region

The cytochrome b(6)f complex: structural studies and comparison with the bc(1) complex.

International audienceElectron crystallography of the chloroplastic b(6)f complex allowed the calculation of projection maps of crystals negatively stained or embedded in glucose. This gives insights into the overall structure of the extra- and transmembrane domains of the complex. A comparison with the structure of the bc(1) complex, the mitochondrial homologue of the b(6)f complex, suggests that the transmembrane domains of the two complexes are very similar, confirming the structural homology deduced from sequence analysis. On the other hand, the extramembrane organisation of the c-type cytochrome and of the Rieske protein seems quite different. Nevertheless, the same type of movement of the Rieske protein is observed in the b(6)f as in the bc(1) complex upon the binding of the quinol analogue stigmatellin. Crystallographic data also suggest movements in the transmembrane domains of the b(6)f complex, which would be specific of the b(6)f complex

Reconnaissance phage-bactérie dans le système phage T5 - E.coli (Caractérisation biochimique et structurale du complexe FhuA-pb5 et de la protéine caudale pb9)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Examining Membrane Proteins by Neutron Scattering

International audienceSmall angle neutron scattering (SANS) is a powerful tool for studying the structure of solubilized membrane proteins. It allows describing the general dimension of the membrane protein , evidence conformational changes, and may provide a low-resolution structure at the nm resolution range. This is because SANS can discriminate between the membrane protein and its amphiphilic partner by specific deuteration of the partners and of the buffer. This chapter was written to offer to a scientist aiming to describe a membrane protein structure the basic tools to consider a SANS experiment. It presents the general principle of contrast variation and a bibliographic survey of experimental strategies used for membrane proteins, some basic theoretical background, and a succinct description of the principles of analysis, of the instrumental and sample requirement, and of the practical steps, prior to the experiments, during the experiments and for data analysis