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Efficient 18.8 T MAS-DNP NMR reveals hidden side chains in amyloid fibrils

Amyloid fibrils are large and insoluble protein assemblies composed of a rigid core associated with a cross- arrangement rich in-sheet structural elements. It has been widely observed in solid-state NMR experiments that semi-rigid protein segments or side chains do not yield easily observable NMR signals at room temperature. The reasons for the missing peaks may be due to the presence of unfavorable dynamics that interfere with NMR experiments, which result in very weak or unobservable NMR signals. Therefore, for amyloid fibrils, semi-rigid and dynamically disordered segments flanking the amyloid core are very challenging to study. Here, we show that high-field dynamic nuclear polarization (DNP), an NMR hyperpolarization technique typically performed at low temperatures, can circumvent this issue because (i) the low-temperature environment (~ 100 K) slows down the protein dynamics to escape unfavorable detection regime, (ii) DNP improves the overall NMR sensitivity including flexible side chains, and (iii) efficient cross-effect DNP biradicals (SNAPol-1) optimized for high-field DNP ( 18.8 T) are employed to offer high sensitivity and resolution suitable for biomolecular NMR applications. By combining these factors, we have successfully recorded an unprecedented enhancement factor of ε~50 on amyloid fibrils using an 18.8 T/ 800 MHz magnet. We have compared the DNP efficiencies of M-TinyPol, NATriPol-3, and SNAPol-1 biradicals on amyloid fibrils. We found that SNAPol-1 (with ε~50) outperformed the other two radicals. The MAS DNP experiments revealed signals of flexible side chains previously inaccessible at conventional room-temperature experiments. These results demonstrate the potential of MAS-DNP NMR as a valuable tool for structural investigations of amyloid fibrils, particularly for side chains and dynamically disordered segments otherwise hidden at room temperature

Efficient high-field MAS-DNP NMR reveals hidden side chains in amyloid fibrils

It has been widely observed in bioNMR experiments that many biological molecules contain flexible parts or side chains that do not yield easily observable NMR signals in room-temperature experiments. The reasons for the missing peaks could be because the flexible regions might exhibit unfavorable dynamics that interfere with NMR experiments, which result in low NMR intensity below the noise floor. To circumvent this issue, we exploit a hyperpolarization technique known as dynamic nuclear polarization (DNP), which is usually performed at low temperatures for optimal performances. We have also compared 1H enhancements for amyloid fibrils doped with the SNAPol-1 and M-Tinypol radicals, and the 1H DNP spectrum demonstrates a much higher ε~30 for the SNAPol-1 radical than ε~10 for the TinyPol-doped sample. By combining the sensitivity gain bestowed by efficient DNP polarizing agent (SNAPol-1), the freezing of local motions at cryogenic temperature (~ 100 K), and high NMR resolution at high magnetic field (18.8 T), we have successfully recorded an unprecedented enhancement factor of ~50 on amyloid fibrils (NWD2) in magic-angle spinning (MAS) DNP experiments at a high magnetic field of 18.8 T. Moreover, multidimensional MAS NMR experiments have revealed NMR signals of flexible side chains that were previously inaccessible at conventional room-temperature experiments. We also demonstrate that sensitivity-enhanced 2D 15N-13C correlation experiments can be achieved in ~ 2 hours. These results demonstrate the potential of MAS-DNP NMR as a valuable tool for structural investigations of amyloid fibrils, particularly for side chains otherwise hidden at room temperature

Supramolecular organisation and dynamics of mannosylated phosphatidylinositol lipids in the mycobacterial plasma membrane

International audienceMycobacterium tuberculosis ( Mtb ) is the causative agent of tuberculosis (TB), a disease that claims ~1.6 million lives annually. The current treatment regime is long and expensive, and missed doses contribute to drug resistance. Therefore, development of new anti-TB drugs remains one of the highest public health priorities. Mtb has evolved a complex cell envelope that represents a formidable barrier to antibiotics. The Mtb cell envelop consists of four distinct layers enriched for Mtb specific lipids and glycans. Although the outer membrane, comprised of mycolic acid esters, has been extensively studied, less is known about the plasma membrane, which also plays a critical role in impacting antibiotic efficacy. The Mtb plasma membrane has a unique lipid composition, with mannosylated phosphatidylinositol lipids (phosphatidyl-myoinositol mannosides, PIMs) comprising more than 50% of the lipids. However, the role of PIMs in the structure and function of the membrane remains elusive. Here, we used multiscale molecular dynamics (MD) simulations to understand the structure-function relationship of the PIM lipid family and decipher how they self-organize to shape the biophysical properties of mycobacterial plasma membranes. We assess both symmetric and asymmetric assemblies of the Mtb plasma membrane and compare this with residue distributions of Mtb integral membrane protein structures. To further validate the model, we tested known anti-TB drugs and demonstrated that our models agree with experimental results. Thus, our work sheds new light on the organization of the mycobacterial plasma membrane. This paves the way for future studies on antibiotic development and understanding Mtb membrane protein function

L'ordinaire des sciences et techniques. Cultures populaires, cultures informelles (1)

International audienceLa présente édition de la Lucarne matérialise la poursuite d’un travail engagé depuis 2013. Il a été réalisé dans le cadre d’un atelier collaboratif d’histoire socioculturelle. L’atelier lui-même est animé par un collectif formé des enseignant∙e∙s et auditeur∙rice∙s des formations du CNAM dédiées à la médiation socioculturelle des sciences et techniques. Certificat de compétences, Licence professionnelle ou Magister s’attèlent ainsi à la réflexion, à la conception et à la réalisation de dispositifs pour la médiation des sciences et techniques en société par le design culturel. C’est dans ce cadre de réalisation pédagogique que sont rassemblés ces textes et documents pour lesquels sont proposés de courtes analyses sur leur fonction de support de diffusion massive dans l’espace public d’une culture populaire et ordinaire des sciences et techniques. Tous ont été rédigés dans le cadre d’un exercice exigé pour la validation d’un enseignement. Si le résultat final peut s’avérer inégalement satisfaisant à l’aune des critères de leurs commanditaires, tous les textes apportent cependant un regard jugé pertinent sur un des aspects des manifestations des sciences et techniques dans leurs rapports à la société au travers de la production et de la consommation culturelle de masse. Quelques-uns ont été un peu remaniés pour la présente édition. Retrouvez-les tous, ainsi que d’autres non publiés dans ce volume, sur le site de la Lucarne : http://ateliercst.hypotheses.org

Mechanisms governing subcompartmentalization of biological membranes

Les membranes présentent une grande variété de lipides et de protéines qui agissent sur la biochimie, le transport et la signalisation. La dynamique et l'organisation des composants membranaires sont régulées dans l'espace et dans le temps pour exécuter des fonctions précises. Notre compréhension des mécanismes moléculaires qui façonnent et régissent la sous-compartimentation membranaire et les sites de contact inter-organiques demeure encore limitée. Ici, nous passons en revue certains mécanismes rapportés impliqués dans la régulation des domaines membranaires des plantes, y compris ceux de la membrane plasmatique, des plastides, des mitochondries et du réticulum endoplasmique. Enfin, nous discutons de plusieurs méthodes de pointe qui permettent aujourd'hui aux chercheurs de déchiffrer l'architecture de ces structures au niveau moléculaire et atomique.Membranes show a tremendous variety of lipids and proteins operating biochemistry, transport and signalling. The dynamics and the organization of membrane constituents are regulated in space and time to execute precise functions. Our understanding of the molecular mechanisms that shape and govern membrane subcompartmentalization and inter-organelle contact sites still remains limited. Here, we review some reported mechanisms implicated in regulating plant membrane domains including those of plasma membrane, plastids, mitochondria and endoplasmic reticulum. Finally, we discuss several state-of-the-art methods that allow nowadays researchers to decipher the architecture of these structures at the molecular and atomic level

Femtosecond X-ray coherent diffraction of aligned amyloid fibrils on low background graphene

Here we present a new approach to diffraction imaging of amyloid fibrils, combining a free-standing graphene support and single nanofocused X-ray pulses of femtosecond duration from an X-ray free-electron laser. Due to the very low background scattering from the graphene support and mutual alignment of filaments, diffraction from tobacco mosaic virus (TMV) filaments and amyloid protofibrils is obtained to 2.7 Å and 2.4 Å resolution in single diffraction patterns, respectively. Some TMV diffraction patterns exhibit asymmetry that indicates the presence of a limited number of axial rotations in the XFEL focus. Signal-to-noise levels from individual diffraction patterns are enhanced using computational alignment and merging, giving patterns that are superior to those obtainable from synchrotron radiation sources. We anticipate that our approach will be a starting point for further investigations into unsolved structures of filaments and other weakly scattering objects

Femtosecond X-ray coherent diffraction of aligned amyloid fibrils on low background graphene

Here we present a new approach to diffraction imaging of amyloid fibrils, combining a freestanding graphene support and single nanofocused X-ray pulses of femtosecond duration from an X-ray free-electron laser. Due to the very low background scattering from the graphene support and mutual alignment of filaments, diffraction from tobacco mosaic virus (TMV) filaments and amyloid protofibrils is obtained to 2.7 A and 2.4 A resolution in single diffraction patterns, respectively. Some TMV diffraction patterns exhibit asymmetry that indicates the presence of a limited number of axial rotations in the XFEL focus. Signal-to-noise levels from individual diffraction patterns are enhanced using computational alignment and merging, giving patterns that are superior to those obtainable from synchrotron radiation sources. We anticipate that our approach will be a starting point for further investigations into unsolved structures of filaments and other weakly scattering objects

L'ordinaire des sciences et techniques. Cultures populaires, cultures informelles (2)

International audienceLa présente édition de la Lucarne matérialise la poursuite d’un travail engagé depuis 2013. Comme pour la précédente livraison, il a été réalisé dans le cadre d’un atelier collaboratif d’histoire socioculturelle. L’atelier lui-même est animé par un collectif formé des enseignant∙e∙s et auditeur∙rice∙s des formations du CNAM dédiées à la médiation socioculturelle des sciences et techniques. Certificat de compétences, Licence professionnelle ou Magister s’attèlent ainsi à la réflexion, à la conception et à la réalisation de dispositifs pour la médiation des sciences et techniques en société par le design culturel. C’est dans ce cadre de réalisation pédagogique que sont rassemblés ces textes et documents pour lesquels sont proposés de courtes analyses sur leur fonction de support de diffusion massive dans l’espace public d’une culture populaire et ordinaire des sciences et techniques. Tous ont été rédigés dans le cadre d’un exercice exigé pour la validation d’un enseignement. Si le résultat final peut s’avérer inégalement satisfaisant à l’aune des critères de leurs commanditaires, tous les textes apportent cependant un regard jugé pertinent sur un des aspects des manifestations des sciences et techniques dans leurs rapports à la société au travers de la production et de la consommation culturelle de masse. Quelques-uns ont été un peu remaniés pour la présente édition. Retrouvez-les tous, ainsi que d’autres non publiés dans ce volume, sur le site de la Lucarne : http://ateliercst.hypotheses.org