Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae

Abstract RecA-mediated Homologous Recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae ( Sp RecA), in comparison to the Escherichia coli RecA ( Ec RecA) paradigm. Ec RecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block Ec RecA nucleofilament growth. We report that neither of the two paralogous pneumococcal SSBs could assist Sp RecA polymerization on ssDNA. Instead, we found that the conserved RadA helicase promotes this Sp RecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native Sp RecA nucleopolymer by cryoEM stabilized with ATPγS. It was found to be equivalent to the crystal structure of the Ec RecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that Sp RecA and Ec RecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology

Single-molecule FRET on its way to structural biology in live cells

International audienceSingle-molecule FRET is making stepwise progress toward the realization of its full potential: becoming the reference technique to monitor protein structural dynamics in live cells

Studying GPCR conformational dynamics by single molecule fluorescence

International audienceOver the last decades, G protein coupled receptors (GPCRs) have experienced a tremendous amount of attention, which has led to a boost of structural and pharmacological insights on this large membrane protein superfamily involved in various essential physiological functions. Recently, evidence has emerged that, rather than being activated by ligands in an on/off manner switching from an inactive to an active state, GPCRs exhibit high structural flexibility in the absence and even in the presence of ligands. So far the physiological as well as pharmacological impact of this structural flexibility remains largely unexplored albeit its potential role in precisely fine-tuning receptor function and regulating the specificity of signal transduction into the cell. By complementing other biophysical approaches, single molecule fluorescence (SMF) offers the advantage of monitoring structural dynamics in biomolecules in real-time, with minimal structural invasiveness and in the context of complex biological environments. In this review a general introduction to GPCR structural dynamics is given followed by a presentation of SMF methods used to explore them. Particular attention is paid to single molecule Förster resonance energy transfer (smFRET), a key method to measure actual distance changes between two probes, and highlight conformational changes occurring at timescales relevant for protein conformational movements. The available literature reporting on GPCR structural dynamics by SMF is discussed with a focus on the newly gained biological insights on receptor activation and signaling, in particular for the β2 adrenergic and the metabotropic glutamate receptors

Energétique et dynamique structurale des interactions des récepteurs des oestrogènes humains

Les récepteurs des estrogènes sont impliqués dans la croissance et la différenciation des tissus reproductifs. Ce sont des facteurs de transcription inductibles qui se lient à des séquences spécifiques d'ADN situées en amont du gène régulé.En réponse à la liaison de molécules agonistes ils interagissent avec des protéines co-activatrices, conduisant par la suite au recrutement de l'ensemble de la machinerie transcriptionnelle. Une compréhension fine des mécanismes d'activation transcriptionnelle par les récepteurs nucléaires en général nécessite, outre des informations structurales, la caractérisation des paramètres énergétiques gouvernant ces interactions macromoléculaires. Nous avons utilisé dans ce travail la spectroscopie de fluorescence pour étudier les interactions in vitro entre les récepteurs des oestrogènes a et b recombinants purifiés, et leur ADN cibles, leur ligands et certains coactivateurs.Nous avons pu mettre en évidence les caractéristiques thermodynamiques de la liaison de ER à un ADN fluorescent. Le rôle énergétique joué par les différentes bases de l' élément de réponse a été quantifié, et le rôle pouvant être joué par les ligands sur la multimérisation du récepteur a été mis en évidence. De plus, nous avons caractérisé les interactions entre ER a et ß entiers ainsi que le domaine de liaison de l'homme isolé ( ERaHBD) d'une part, et des fragments des cofacteurs TIF1a , SRC-1 et TIF-2 d'autre part. Les expériences d'anisotropie de fluorescence et de spectroscopie de corrélation de fluorescence sur molécule unique conduites à l'aide du fragment SRC-1570-780 marqué par un fluorophore ont démontré sans ambiguïté une stœchiométrie de 1 molécule de SRC-1 par dimère de Era. Nous avons également déterminé l'affinité de ces interactions en présence de différents agoniste, et nous avons pu montrer l'importance de la structure de ceux-ci sur l'affinité d'interaction, indépendamment de leurs propres affinités pour le récepteur.MONTPELLIER-BU Pharmacie (341722105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Synchronous, Crosstalk-free Correlative AFM and Confocal Microscopies/Spectroscopies

International audienceMicroscopies have become pillars of our characterization tools to observe biological systems and assemblies. Correlative and synchronous use of different microscopies relies on the fundamental assumption of non-interference during images acquisitions. In this work, by exploring the correlative use of Atomic Force Microscopy and confocal-Fluorescence-Lifetime Imaging Microscopy (AFM-FLIM), we quantify cross-talk effects occurring during synchronous acquisition. We characterize and minimize optomechanical forces on different AFM cantilevers interfering with normal AFM operation as well as spurious luminescence from the tip and cantilever affecting time-resolved fluorescence detection. By defining non-interfering experimental imaging parameters, we show accurate real-time acquisition and two-dimensional mapping of interaction force, fluorescence lifetime and intensity characterizing morphology (AFM) and local viscosity (FLIM) of gel and fluid phases separation of supported lipid model membranes. Finally, as proof of principle by means of synchronous force and fluorescence spectroscopies, we precisely tune the lifetime of a fluorescent nanodiamond positioned on the AFM tip by controlling its distance from a metallic surface. This opens up a novel pathway of quench sensing to image soft biological samples such as membranes since it does not require tip-sample mechanical contact in contrast with conventional AFM in liquid