Structural study of the membrane protein MscL using cell-free expression and solid-state

a b s t r a c t High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75 kDa pentameric a-helical ion channel from Escherichia coli, reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state NMR

NOEnet–Use of NOE networks for NMR resonance assignment of proteins with known 3D structure

Motivation: A prerequisite for any protein study by NMR is the assignment of the resonances from the 15N−1H HSQC spectrum to their corresponding atoms of the protein backbone. Usually, this assignment is obtained by analyzing triple resonance NMR experiments. An alternative assignment strategy exploits the information given by an already available 3D structure of the same or a homologous protein. Up to now, the algorithms that have been developed around the structure-based assignment strategy have the important drawbacks that they cannot guarantee a high assignment accuracy near to 100%

Robust structure-based resonance assignment for functional protein studies by NMR

High-throughput functional protein NMR studies, like protein interactions or dynamics, require an automated approach for the assignment of the protein backbone. With the availability of a growing number of protein 3D structures, a new class of automated approaches, called structure-based assignment, has been developed quite recently. Structure-based approaches use primarily NMR input data that are not based on J-coupling and for which connections between residues are not limited by through bonds magnetization transfer efficiency. We present here a robust structure-based assignment approach using mainly HN–HN NOEs networks, as well as 1H–15N residual dipolar couplings and chemical shifts. The NOEnet complete search algorithm is robust against assignment errors, even for sparse input data. Instead of a unique and partly erroneous assignment solution, an optimal assignment ensemble with an accuracy equal or near to 100% is given by NOEnet. We show that even low precision assignment ensembles give enough information for functional studies, like modeling of protein-complexes. Finally, the combination of NOEnet with a low number of ambiguous J-coupling sequential connectivities yields a high precision assignment ensemble. NOEnet will be available under: http://www.icsn.cnrs-gif.fr/download/nmr

Infranalytics : une plateforme nationale pour accéder aux spectromètres les plus puissants

National audienc

Infranalytics : une plateforme nationale pour accéder aux spectromètres les plus puissants

National audienc

Etude par RMN de protéines et de peptides régulateurs de la poymérisation de l'actine

Les protéines à domaine WH2/thymosine b, notamment la thymosine b4 et le domaine l de ciboulot, jouent un rôle important dans le mécanisme de régulation de la polymérisation de l actine. La thymosine b4 inhibe la formation des filaments de l'actine par la séquestration des monomères. A l'inverse, ciboulot D1 participe à la polymérisation de l'actine. Une série de protéines chimères de la thymosine b4 et du premier domaine de ciboulot ont été construites et étudiées afin d'évaluer l'influence de chaque région des domaines WH2/Tb (N-terminale, linker, motif consensus et C-terminale) sur leurs interactions avec l'actine et d'identifier les déterminants des fonctions séquestrantes ou promotrices de la formation du filament. Les complexes des protéines à domaine WH2/Tb avec l'actine ont été caractérisés de manière détaillée d'un point de vue structural et dynamique. Ces études ont permis d'affiner le modèle fonctionnel, de mettre en évidence des éléments importants de l'interaction de ces protéines avec l'actine et d'analyser le comportement des différentes protéines selon leur fonction. L'influence de peptides vecteurs amphiphiles et/ou chargés positivement sur la polymérisation de l'actine a été abordée par RMN. Leur liaison au monomère d'actine et la formation induite de particules de haute masse ont pu être mises en évidence. En parallèle d'expériences de couplage chimique, le déplacement de la thymosine 4 a permis de proposer un site d'interaction pour ces peptides. Ces peptides ne peuvent donc plus être considérés comme de simples vecteurs passifs et leur rôle propre dans la restauration du filament ouvre d'intéressantes perspectives.bthymosin and WH2 domains are widespread, intrinsically disordered actin-binding peptides which display versatile regulation of actin assembly in motile processes. Thymosin-b4 sequesters G-actin, whereas the complex of G-actin with the first b-thymosin domain of Ciboulot or conventional WH2 domains participates in filament barbed end assembly like profilin. Here, using chimeras of ciboulot and Thymosin-b4, we show the role of their different regions for their binding and function with actin and the regulation of the dynamics in tbeir 1:1 complexes with G-actin at physiological ionic strength. A single salt bridge with actin located in Thymosin-b4 linker before its LKKT/V motif stabilizes aIl its central and C-terminal interactions and induces sequestration when inserted in Ciboulot. NMR relaxation structural studies are combined on CibD1/T b 4 chimeras to show that the central and C-terminal regions of cibD1 and Tb4 display an equivalent G-actin sequestering capacity and that the sequestering and profilin-like activities of both bT and long WH2 dornains are regulated at physiological ionic strength by dynamic interactions in their stoichiometric 1:1 complexes with G-actin. Cell-penetrating peptides can cross cell membranes and are commonly seen as biologically inert molecules. However, we found that some CPP could remodel actin cytoskeleton. These arginine- and/or tryptophan-rich peptides could cross cell membrane and induced stress fibers formation. The peptides could bind directly monomeric actin as determined by NMR and calorimetry studies. Therefore, cell-penetrating peptides might interact with intracellular protein partners, such as actin.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Spatially encoded 2D and 3D diffusion-ordered NMR spectroscopy

We show that the acquisition of 3D diffusion-ordered NMR spectroscopy (DOSY) experiments can be accelerated significantly with the use of spatial encoding (SPEN). The SPEN DOSY approach is discussed, analysed with numerical simulation, and illustrated on a mixture of small molecules