Il centro di reazione fotosintetico batterico in ambiente nativo ed artificiale: effetti sul trasferimento elettronico

In this thesis we focussed on the characterization of the reaction center (RC) protein purified from the photosynthetic bacterium Rhodobacter sphaeroides. In particular, we discussed the effects of native and artificial environment on the light-induced electron transfer processes. The native environment consist of the inner antenna LH1 complex that copurifies with the RC forming the so called core complex, and the lipid phase tightly associated with it. In parallel, we analyzed the role of saccharidic glassy matrices on the interplay between electron transfer processes and internal protein dynamics. As a different artificial matrix, we incorporated the RC protein in a layer-by-layer structure with a twofold aim: to check the behaviour of the protein in such an unusual environment and to test the response of the system to herbicides. By examining the RC in its native environment, we found that the light-induced charge separated state P+QB - is markedly stabilized (by about 40 meV) in the core complex as compared to the RC-only system over a physiological pH range. We also verified that, as compared to the average composition of the membrane, the core complex copurifies with a tightly bound lipid complement of about 90 phospholipid molecules per RC, which is strongly enriched in cardiolipin. In parallel, a large ubiquinone pool was found in association with the core complex, giving rise to a quinone concentration about ten times larger than the average one in the membrane. Moreover, this quinone pool is fully functional, i.e. it is promptly available at the QB site during multiple turnover excitation of the RC. The latter two observations suggest important heterogeneities and anisotropies in the native membranes which can in principle account for the stabilization of the charge separated state in the core complex. The thermodynamic and kinetic parameters obtained in the RC-LH1 complex are very close to those measured in intact membranes, indicating that the electron transfer properties of the RC in vivo are essentially determined by its local environment. The studies performed by incorporating the RC into saccharidic matrices evidenced the relevance of solvent-protein interactions and dynamical coupling in determining the kinetics of electron transfer processes. The usual approach when studying the interplay between internal motions and protein function consists in freezing the degrees of freedom of the protein at cryogenic temperature. We proved that the “trehalose approach” offers distinct advantages with respect to this traditional methodology. We showed, in fact, that the RC conformational dynamics, coupled to specific electron transfer processes, can be modulated by varying the hydration level of the trehalose matrix at room temperature, thus allowing to disentangle solvent from temperature effects. The comparison between different saccharidic matrices has revealed that the structural and dynamical protein-matrix coupling depends strongly upon the sugar. The analyses performed in RCs embedded in polyelectrolyte multilayers (PEM) structures have shown that the electron transfer from QA - to QB, a conformationally gated process extremely sensitive to the RC environment, can be strongly modulated by the hydration level of the matrix, confirming analogous results obtained for this electron transfer reaction in sugar matrices. We found that PEM-RCs are a very stable system, particularly suitable to study the thermodynamics and kinetics of herbicide binding to the QB site. These features make PEM-RC structures quite promising in the development of herbicide biosensors. The studies discussed in the present thesis have shown that, although the effects on electron transfer induced by the native and artificial environments tested are markedly different, they can be described on the basis of a common kinetic model which takes into account the static conformational heterogeneity of the RC and the interconversion between conformational substates. Interestingly, the same distribution of rate constants (i.e. a Gamma distribution function) can describe charge recombination processes in solutions of purified RC, in RC-LH1 complexes, in wet and dry RC-PEM structures and in glassy saccharidic matrices over a wide range of hydration levels. In conclusion, the results obtained for RCs in different physico-chemical environments emphasize the relevance of the structure/dynamics solvent/protein coupling in determining the energetics and the kinetics of electron transfer processes in a membrane protein complex

<em>In vitro</em> Investigation of the MexAB Efflux Pump From <em>Pseudomonas aeruginosa</em>

International audienceThere is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram negative bacteria Pseudomonas aeruginosa in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled: compartmentation in a lipidic environment, use of a relevant index for transport and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient

La cryo-microscopie électronique révèle une nouvelle vision de la cellule et de ses composants

La cryo-microscopie électronique (cryo-EM) est une technique d’imagerie du vivant qui prend désormais une place prépondérante en biologie structurale, avec des retombées en biologie cellulaire et du développement, en bioinformatique, en biomédecine ou en physique de la cellule. Elle permet de déterminer des structures de protéines purifiées in vitro ou au sein des cellules. Cette revue décrit les principales avancées récentes de la cryo-EM, illustrées par des exemples d’élucidation de structures de protéines d’intérêt en biomédecine, et les pistes de développements futurs

In vitro transport activity of the fully assembled MexAB-OprM efflux pump from Pseudomonas aeruginosa

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C: Cryo-Electron Microscopy of Membrane Contact Sites and Their Components

Electron microscopy has played a pivotal role in elucidating the ultrastructure of membrane contact sites between cellular organelles. The advent of cryo-electron microscopy has ushered in the ability to determine atomic models of constituent proteins or protein complexes within sites of membrane contact through single particle analysis. Furthermore, it enables the visualization of the three-dimensional architecture of membrane contact sites, encompassing numerous copies of proteins, whether in vitro reconstituted or directly observed in situ using cryo-electron tomography. Nevertheless, there exists a scarcity of cryo-electron microscopy studies focused on the site of membrane contact and their constitutive proteins. This review provides an overview of the contributions made by cryo-electron microscopy to our understanding of membrane contact sites, outlines the associated limitations, and explores prospects in this field

An Intrinsically Disordered Region in OSBP Acts as an Entropic Barrier to Control Protein Dynamics and Orientation at Membrane Contact Sites

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Caveolin-Assisted Sphingolipid Transport to the Plasma Membrane

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Binding, reconstitution and 2D crystallization of membrane or soluble proteins onto functionalized lipid layer observed in situ by reflected light microscopy

Monolayer of functionalized lipid spread at the air/water interface is used for the structural analysis of soluble and membrane proteins by electron crystallography and single particle analysis. This powerful approach lacks of a method for the screening of the binding of proteins to the surface of the lipid layer. Here, we described an optical method based on the use of reflected light microscopy to image, without the use of any labeling, the lipid layer at the surface of buffers in the Teflon wells used for 2D crystallization. Images revealed that the lipid layer was made of a monolayer coexisting with liposomes or aggregates of lipids floating at the surface. Protein binding led to an increase of the contrast and the decrease of the fluidity of the lipid surface, as demonstrated with the binding of soluble Shiga toxin B subunit, of purple membrane and of solubilized His-BmrA, a bacterial ABC transporter. Moreover the reconstitution of membrane proteins bound to the lipidic surface upon detergent removal can be followed through the appearance of large recognizable domains at the surface. Proteins binding and reconstitution were further confirmed by electron microcopy. Overall, this method provides a quick evaluation of the monolayer trials, a significant reduction in screening by transmission electron microscopy and new insights in the proteins binding and 2D crystallogenesis at the lipid surface