19 research outputs found
Sensitive proton-detected solid-state NMR spectroscopy of large proteins with selective CH3 labelling: application to the 50S ribosome subunit
International audienceSolid-state NMR spectroscopy allows the characterization of structure, interactions and dynamics of insoluble and/or very large proteins. Sensitivity and resolution are often major challenges for obtaining atomic-resolution information, in particular for very large protein complexes. Here we show that the use of deuterated, specifically CH3-labelled proteins result in significant sensitivity gains compared to previously employed CHD2 labelling, while line widths only marginally increase. We apply this labelling strategy to a 468 kDa-large dodecameric aminopeptidase, TET2, and the 1.6 MDa-large 50S ribosome subunit of Thermus thermophilus
How Detergent Impacts Membrane Proteins: Atomic-Level Views of Mitochondrial Carriers in Dodecylphosphocholine.
Characterizing the structure of membrane proteins (MPs) generally requires extraction from their native environment, most commonly with detergents. Yet, the physicochemical properties of detergent micelles and lipid bilayers differ markedly and could alter the structural organization of MPs, albeit without general rules. Dodecylphosphocholine (DPC) is the most widely used detergent for MP structure determination by NMR, but the physiological relevance of several prominent structures has been questioned, though indirectly, by other biophysical techniques, e.g., functional/thermostability assay (TSA) and molecular dynamics (MD) simulations. Here, we resolve unambiguously this controversy by probing the functional relevance of three different mitochondrial carriers (MCs) in DPC at the atomic level, using an exhaustive set of solution-NMR experiments, complemented by functional/TSA and MD data. Our results provide atomic-level insight into the structure, substrate interaction and dynamics of the detergent-membrane protein complexes and demonstrates cogently that, while high-resolution NMR signals can be obtained for MCs in DPC, they systematically correspond to nonfunctional states
Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development
12 pags., 4 figs., 3 tabs.SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80â% of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.Work at BMRZ is supported by the state of Hesse. Work in Covid19-NMR
was supported by the Goethe Corona Funds, by the IWBEFRE-program 20007375 of state of Hesse, the DFG
through CRC902: âMolecular Principles of RNA-based regulation.â and through infrastructure funds (project
numbers: 277478796, 277479031, 392682309, 452632086, 70653611) and by European Unionâs Horizon 2020 research and innovation program iNEXT-discovery under grant agreement No 871037. BY-COVID receives funding from the European Unionâs Horizon Europe Research and Innovation Programme under grant agreement number 101046203. âINSPIREDâ (MIS 5002550) project, implemented under the Action âReinforcement of the Research and Innovation Infrastructure,â funded by the Operational
Program âCompetitiveness, Entrepreneurship and Innovationâ (NSRF 2014â2020) and co-financed by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011-285950ââSEE-DRUGâ project (purchase of UPATâs 700 MHz NMR equipment). The support of the CERM/CIRMMP center of Instruct-ERIC is gratefully acknowledged. This work has been funded in part by a grant of the Italian Ministry of University and Research (FISR2020IP_02112, ID-COVID) and by Fondazione CR
Firenze. A.S. is supported by the Deutsche Forschungsgemeinschaft [SFB902/B16, SCHL2062/2-1] and the Johanna Quandt Young Academy at Goethe [2019/AS01]. M.H. and C.F. thank SFB902 and the Stiftung Polytechnische Gesellschaft for the Scholarship. L.L. work was supported by the French National Research Agency (ANR, NMR-SCoV2-ORF8), the Fondation de la Recherche MĂ©dicale (FRM, NMR-SCoV2-ORF8), FINOVI and the IR-RMN-THC Fr3050 CNRS. Work at UConn Health was supported by grants from the US National Institutes of Health (R01 GM135592 to B.H., P41 GM111135 and R01 GM123249 to J.C.H.) and the US National Science Foundation (DBI 2030601 to J.C.H.). Latvian Council of Science Grant No. VPP-COVID-2020/1-0014. National Science Foundation EAGER MCB-2031269. This work was supported by the grant Krebsliga KFS-4903-08-2019 and SNF-311030_192646 to J.O. P.G. (ITMP) The EOSC Future project is co-funded by the European Union Horizon Programme call INFRAEOSC-03-2020âGrant Agreement
Number 101017536. Open Access funding enabled and organized by Projekt DEALPeer reviewe
Fonction d'une protéine membranaire : étude structurale et dynamique par RMN
The use of detergents is often unavoidable in the structural studies of membrane proteins. Dodecylphosphocholine (DPC) is one of the most commonly used detergents for such studies in solution state NMR spectroscopy. The effect of detergent on structure and dynamics remains an important and poorly understood question. In this study we have investigated millisecond dynamics, substrate binding and structural features of three different yeast proteins from mitochondrial carrier family (GGC1, ORC1 and AAC3) in DPC micelles. We have detected millisecond dynamics, which are asymmetrically distributed across the structure. Contrary to previous claims, we show that these dynamics are unrelated to function, as they are not affected by the substitutions which abolish mitochondrial carrier transport in proteoliposomes. Furthermore, we could show that the very well-defined substrate specificity of these proteins in membranes is abolished when they are reconstituted in DPC, questioning their functionality. Structural investigations have revealed that both tertiary and secondary structures of these carriers are perturbed in DPC micelles, with some TM helices showing substantial solvent exposure. We have concluded from these observations that DPC detergent strongly perturbs these, and likely other mitochondrial carriers by rendering them very flexible. Our findings point to a possibly general effect of this detergent on membrane proteins, as we discuss with examples of previously studied membrane proteins. In the second part we have addressed a fundamental question of protein dynamics: how do proteins move inside crystals? We have investigated ms dynamics in a crystalline ubiquitin to gain the insight on the impact of the crystalline lattice on such motions, using solid-state NMR and ms long MD simulations of explicit crystal arrangements. Interestingly a local dynamic exchange process on a ms time scale is still present in crystals. However, by comparing different crystal forms we establish that the thermodynamics of the exchanging states and their interconversion rate constants are significantly altered by the crystal contacts. Furthermore, we detect overall "rocking" motion of molecules in the crystal, occurring on a tens-of-ms time scale, and provide evidence that overall and local motion are coupled. We discuss the implications of ms dynamics on the data quality in X-ray diffraction experiments.Lâutilisation de dĂ©tergents est inĂ©vitable pour les Ă©tudes structurales des protĂ©ines membranaires. Dodecylphosphocholine (DPC) est un des dĂ©tergents les plus utilisĂ©s pour ce type dâĂ©tudes employant la spectroscopie de rĂ©sonance magnĂ©tique nuclĂ©aire (RMN) en solution. Lâeffet des dĂ©tergents sur la structure et la dynamique des macromolĂ©cules est une problĂ©matique importante, mais peu Ă©tudiĂ©e Ă ce jour. Dans cette Ă©tude nous avons caractĂ©risĂ© la dynamique Ă lâĂ©chelle de la milliseconde, la liaison des substrats ainsi que des propriĂ©tĂ©s structurales de trois protĂ©ines membranaires diffĂ©rentes solubilisĂ©es dans des micelles de DPC. Ces protĂ©ines font partie de la famille des transporteurs mitochondriaux et nous avons choisi les sĂ©quences de la levure (ORC1, GGC1, AAC3). Nous avons dĂ©tectĂ© de la dynamique Ă lâĂ©chelle de la milliseconde qui est distribuĂ©e dâune maniĂšre asymĂ©trique Ă travers la structure. En contradiction avec des propos de la littĂ©rature, nous montrons que cette dynamique nâest pas corrĂ©lĂ©e Ă la fonction, puisquâelle nâest pas modifiĂ©e par des mutations qui inhibent le transport effectuĂ© par ces protĂ©ines quand elles sont reconstituĂ©es dans des liposomes. En plus, nous avons pu montrer que leur spĂ©cificitĂ© par rapport aux substrats, nâest pas conservĂ©e quand ces transporteurs sont reconstituĂ©s dans du DPC, mettant en question leur fonctionnalitĂ© dans ce dĂ©tergent. La RMN a aussi permis de dĂ©montrer que les structures tertiaire et secondaire sont perturbĂ©es dans les micelles avec quelques hĂ©lices transmembranaires apparaissant exposĂ©es au solvant. Nous avons donc conclu que la prĂ©sence du dĂ©tergent a un effet fort sur les trois transporteurs mitochondriaux de notre Ă©tude et probablement dâautres protĂ©ines similaires, en les rendant trĂšs flexible. Nos rĂ©sultats indiquent un probable effet gĂ©nĂ©ral de ce dĂ©tergent sur les protĂ©ines membranaires, comme nous le discutons dans une analyse dĂ©taillĂ©e de quelques Ă©tudes de protĂ©ines membranaires dĂ©crites dans la littĂ©rature. Dans la seconde partie de ce travail, nous avons adressĂ© une question fondamentale de la dynamique des protĂ©ines: comment se comportent les protĂ©ines dans des cristaux ? Nous avons Ă©tudiĂ© la dynamique de lâubiquitine cristalline Ă lâĂ©chelle de la milliseconde afin de comprendre lâinfluence de la maille cristalline sur ce type de mouvement. Pour ce faire, nous avons employĂ© la RMN Ă lâĂ©tat solide et des simulations de dynamique molĂ©culaire de la protĂ©ine dans diffĂ©rents rĂ©seaux cristallins distincts. Il est intĂ©ressant Ă noter que dans ces cristaux on dĂ©tecte toujours des processus locaux dâĂ©change dynamique sur une Ă©chelle de temps de la milliseconde. Cependant, en comparant les rĂ©sultats obtenus avec diffĂ©rentes formes cristallines, nous constatons que les paramĂštres thermodynamiques des diffĂ©rents Ă©tats en Ă©change et les vitesses dâinterconversion entre ces derniĂšres sont significativement modifiĂ©s par les contacts cristallins. De plus, nous avons dĂ©tectĂ© des mouvements globaux de type «rocking» des ces molĂ©cules Ă lâĂ©tat cristallin qui surviennent Ă©galement Ă lâĂ©chelle de la milliseconde. Ceci suggĂšre que les mouvements globaux et locaux sont corrĂ©lĂ©s. Cette observation ouvre la discussion de lâimportance de ce type de mouvements pour la qualitĂ© et lâinterprĂ©tation des donnĂ©es des expĂ©riences de diffraction des rayons-X
The function of a membrane protein : studies of structure and dynamics by NMR
Lâutilisation de dĂ©tergents est inĂ©vitable pour les Ă©tudes structurales des protĂ©ines membranaires. Dodecylphosphocholine (DPC) est un des dĂ©tergents les plus utilisĂ©s pour ce type dâĂ©tudes employant la spectroscopie de rĂ©sonance magnĂ©tique nuclĂ©aire (RMN) en solution. Lâeffet des dĂ©tergents sur la structure et la dynamique des macromolĂ©cules est une problĂ©matique importante, mais peu Ă©tudiĂ©e Ă ce jour. Dans cette Ă©tude nous avons caractĂ©risĂ© la dynamique Ă lâĂ©chelle de la milliseconde, la liaison des substrats ainsi que des propriĂ©tĂ©s structurales de trois protĂ©ines membranaires diffĂ©rentes solubilisĂ©es dans des micelles de DPC. Ces protĂ©ines font partie de la famille des transporteurs mitochondriaux et nous avons choisi les sĂ©quences de la levure (ORC1, GGC1, AAC3). Nous avons dĂ©tectĂ© de la dynamique Ă lâĂ©chelle de la milliseconde qui est distribuĂ©e dâune maniĂšre asymĂ©trique Ă travers la structure. En contradiction avec des propos de la littĂ©rature, nous montrons que cette dynamique nâest pas corrĂ©lĂ©e Ă la fonction, puisquâelle nâest pas modifiĂ©e par des mutations qui inhibent le transport effectuĂ© par ces protĂ©ines quand elles sont reconstituĂ©es dans des liposomes. En plus, nous avons pu montrer que leur spĂ©cificitĂ© par rapport aux substrats, nâest pas conservĂ©e quand ces transporteurs sont reconstituĂ©s dans du DPC, mettant en question leur fonctionnalitĂ© dans ce dĂ©tergent. La RMN a aussi permis de dĂ©montrer que les structures tertiaire et secondaire sont perturbĂ©es dans les micelles avec quelques hĂ©lices transmembranaires apparaissant exposĂ©es au solvant. Nous avons donc conclu que la prĂ©sence du dĂ©tergent a un effet fort sur les trois transporteurs mitochondriaux de notre Ă©tude et probablement dâautres protĂ©ines similaires, en les rendant trĂšs flexible. Nos rĂ©sultats indiquent un probable effet gĂ©nĂ©ral de ce dĂ©tergent sur les protĂ©ines membranaires, comme nous le discutons dans une analyse dĂ©taillĂ©e de quelques Ă©tudes de protĂ©ines membranaires dĂ©crites dans la littĂ©rature. Dans la seconde partie de ce travail, nous avons adressĂ© une question fondamentale de la dynamique des protĂ©ines: comment se comportent les protĂ©ines dans des cristaux ? Nous avons Ă©tudiĂ© la dynamique de lâubiquitine cristalline Ă lâĂ©chelle de la milliseconde afin de comprendre lâinfluence de la maille cristalline sur ce type de mouvement. Pour ce faire, nous avons employĂ© la RMN Ă lâĂ©tat solide et des simulations de dynamique molĂ©culaire de la protĂ©ine dans diffĂ©rents rĂ©seaux cristallins distincts. Il est intĂ©ressant Ă noter que dans ces cristaux on dĂ©tecte toujours des processus locaux dâĂ©change dynamique sur une Ă©chelle de temps de la milliseconde. Cependant, en comparant les rĂ©sultats obtenus avec diffĂ©rentes formes cristallines, nous constatons que les paramĂštres thermodynamiques des diffĂ©rents Ă©tats en Ă©change et les vitesses dâinterconversion entre ces derniĂšres sont significativement modifiĂ©s par les contacts cristallins. De plus, nous avons dĂ©tectĂ© des mouvements globaux de type «rocking» des ces molĂ©cules Ă lâĂ©tat cristallin qui surviennent Ă©galement Ă lâĂ©chelle de la milliseconde. Ceci suggĂšre que les mouvements globaux et locaux sont corrĂ©lĂ©s. Cette observation ouvre la discussion de lâimportance de ce type de mouvements pour la qualitĂ© et lâinterprĂ©tation des donnĂ©es des expĂ©riences de diffraction des rayons-X.The use of detergents is often unavoidable in the structural studies of membrane proteins. Dodecylphosphocholine (DPC) is one of the most commonly used detergents for such studies in solution state NMR spectroscopy. The effect of detergent on structure and dynamics remains an important and poorly understood question. In this study we have investigated millisecond dynamics, substrate binding and structural features of three different yeast proteins from mitochondrial carrier family (GGC1, ORC1 and AAC3) in DPC micelles. We have detected millisecond dynamics, which are asymmetrically distributed across the structure. Contrary to previous claims, we show that these dynamics are unrelated to function, as they are not affected by the substitutions which abolish mitochondrial carrier transport in proteoliposomes. Furthermore, we could show that the very well-defined substrate specificity of these proteins in membranes is abolished when they are reconstituted in DPC, questioning their functionality. Structural investigations have revealed that both tertiary and secondary structures of these carriers are perturbed in DPC micelles, with some TM helices showing substantial solvent exposure. We have concluded from these observations that DPC detergent strongly perturbs these, and likely other mitochondrial carriers by rendering them very flexible. Our findings point to a possibly general effect of this detergent on membrane proteins, as we discuss with examples of previously studied membrane proteins. In the second part we have addressed a fundamental question of protein dynamics: how do proteins move inside crystals? We have investigated ms dynamics in a crystalline ubiquitin to gain the insight on the impact of the crystalline lattice on such motions, using solid-state NMR and ms long MD simulations of explicit crystal arrangements. Interestingly a local dynamic exchange process on a ms time scale is still present in crystals. However, by comparing different crystal forms we establish that the thermodynamics of the exchanging states and their interconversion rate constants are significantly altered by the crystal contacts. Furthermore, we detect overall "rocking" motion of molecules in the crystal, occurring on a tens-of-ms time scale, and provide evidence that overall and local motion are coupled. We discuss the implications of ms dynamics on the data quality in X-ray diffraction experiments
Protein conformational dynamics studied by (15)N and (1)H R1Ï relaxation dispersion: Application to wild-type and G53A ubiquitin crystals.
International audienceSolid-state NMR spectroscopy can provide site-resolved information about protein dynamics over many time scales. Here we combine protein deuteration, fast magic-angle spinning (~45-60kHz) and proton detection to study dynamics of ubiquitin in microcrystals, and in particular a mutant in a region that undergoes microsecond motions in a ÎČ-turn region in the wild-type protein. We use (15)N R1Ï relaxation measurements as a function of the radio-frequency (RF) field strength, i.e. relaxation dispersion, to probe how the G53A mutation alters these dynamics. We report a population-inversion of conformational states: the conformation that in the wild-type protein is populated only sparsely becomes the predominant state. We furthermore explore the potential to use amide-(1)H R1Ï relaxation to obtain insight into dynamics. We show that while quantitative interpretation of (1)H relaxation remains beyond reach under the experimental conditions, due to coherent contributions to decay, one may extract qualitative information about flexibility
Cross-Correlated Relaxation of Dipolar Coupling and Chemical-Shift Anisotropy in Magic-Angle Spinning R 1Ï NMR Measurements: Application to Protein Backbone Dynamics Measurements
International audienceTransverse relaxation rate measurements in MAS solid-state NMR provide information about molecular motions occurring on nanoseconds-to-milliseconds (ns-ms) time scales. The measurement of heteronuclear (13C , 15N) relaxation rate constants in the presence of a spin-lock radio-frequency field (R1Ï relaxation) provides access to such motions, and an increasing number of studies involving R1Ï relaxation in proteins has been reported. However, two factors that influence the observed relaxation rate constants have so far been neglected, namely (i) the role of CSA/dipolar cross-correlated relaxation (CCR), and (ii) the impact of fast proton spin flips (i.e. proton spin diffusion and relaxation). We show that CSA/D CCR in R1Ï experiments is measurable, and that this cross-correlated relaxation rate constant depends on ns-ms motions, and can thus itself provide insight into dynamics. We find that proton spin-diffusion attenuates this cross-correlated relaxation, due to its decoupling effect on the doublet components. For measurements of dynamics, the use of R1Ï rate constants has practical advantages over the use of CCR rates, and the present manuscript reveals factors that have so far been disregarded and which are important for accurate measurements and interpretation
Cross-Correlated Relaxation of Dipolar Coupling and Chemical-Shift Anisotropy in Magic-Angle Spinning R1 NMR Measurements: Application to Protein Backbone Dynamics Measurements
A ring-shaped conduit connects the mother cell and forespore during sporulation in Bacillus subtilis
During spore formation in Bacillus subtilis a transenvelope complex is assembled across the double membrane that separates the mother cell and forespore. This complex (called the "A-Q complex") is required to maintain forespore development and is composed of proteins with remote homology to components of type II, III, and IV secretion systems found in Gram-negative bacteria. Here, we show that one of these proteins, SpoIIIAG, which has remote homology to ring-forming proteins found in type III secretion systems, assembles into an oligomeric ring in the periplasmic-like space between the two membranes. Three-dimensional reconstruction of images generated by cryo-electron microscopy indicates that the SpoIIIAG ring has a cup-and-saucer architecture with a 6-nm central pore. Structural modeling of SpoIIIAG generated a 24-member ring with dimensions similar to those of the EM-derived saucer. Point mutations in the predicted oligomeric interface disrupted ring formation in vitro and impaired forespore gene expression and efficient spore formation in vivo. Taken together, our data provide strong support for the model in which the A-Q transenvelope complex contains a conduit that connects the mother cell and forespore. We propose that a set of stacked rings spans the intermembrane space, as has been found for type III secretion systems