3D Structure Determination of the Crh Protein from Highly Ambiguous Solid-State NMR Restraints Export

@article{citeulike:3385804, abstract = {Abstract: In a wide variety of proteins, insolubility presents a challenge to structural biology, as X-ray crystallography and liquid-state NMR are unsuitable. Indeed, no general approach is available as of today for studying the three-dimensional structures of membrane proteins and protein fibrils. We here demonstrate, at the example of the microcrystalline model protein Crh, how high-resolution 3D structures can be derived from magic-angle spinning solid-state NMR distance restraints for fully labeled protein samples. First, we show that proton-mediated rare-spin correlation spectra, as well as carbon-13 spin diffusion experiments, provide enough short, medium, and long-range structural restraints to obtain high-resolution structures of this 2 × 10.4 kDa dimeric protein. Nevertheless, the large number of 13C/15N spins present in this protein, combined with solid-state NMR line widths of about 0.5-1 ppm, induces substantial ambiguities in resonance assignments, preventing 3D structure determination by using distance restraints uniquely assigned on the basis of their chemical shifts. In the second part, we thus demonstrate that an automated iterative assignment algorithm implemented in a dedicated solid-state NMR version of the program ARIA permits to resolve the majority of ambiguities and to calculate a de novo 3D structure from highly ambiguous solid-state NMR data, using a unique fully labeled protein sample. We present, using distance restraints obtained through the iterative assignment process, as well as dihedral angle restraints predicted from chemical shifts, the 3D structure of the fully labeled Crh dimer refined at a root-mean-square deviation of 1.33 \r{A}.}, address = {Institut de Biologie et Chimie des Prot\'{e}ines, UMR 5086 CNRS Universit\'{e} Lyon 1, IFR128 BioSciences Lyon-Gerland, 7, passage du Vercors, 69367 Lyon, France, Unit\'{e} de Bio-Informatique Structurale, URA 2185 CNRS, Institut Pasteur, 25-28 rue du docteur Roux, F-75015 Paris, France, and Department of NMR-based Structural Biology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 G\"{o}ttingen, Germany}, author = {Loquet, A. and Bardiaux, B. and Gardiennet, C. and Blanchet, C. and Baldus, M. and Nilges, M. and Malliavin, T. and Bockmann, A.}, citeulike-article-id = {3385804}, citeulike-linkout-0 = {http://dx.doi.org/10.1021/ja078014t}, citeulike-linkout-1 = {http://pubs.acs.org/doi/abs/10.1021/ja078014t}, day = {19}, doi = {10.1021/ja078014t}, journal = {J. Am. Chem. Soc.}, keywords = {crh, ssnmr}, month = {March}, number = {11}, pages = {3579--3589}, posted-at = {2008-10-08 10:52:59}, priority = {2}, title = {3D Structure Determination of the Crh Protein from Highly Ambiguous Solid-State NMR Restraints}, url = {http://dx.doi.org/10.1021/ja078014t}, volume = {130}, year = {2008} }

TY - JOUR ID - citeulike:3385804 L3 - citeulike-article-id:3385804 N2 - Abstract: In a wide variety of proteins, insolubility presents a challenge to structural biology, as X-ray crystallography and liquid-state NMR are unsuitable. Indeed, no general approach is available as of today for studying the three-dimensional structures of membrane proteins and protein fibrils. We here demonstrate, at the example of the microcrystalline model protein Crh, how high-resolution 3D structures can be derived from magic-angle spinning solid-state NMR distance restraints for fully labeled protein samples. First, we show that proton-mediated rare-spin correlation spectra, as well as carbon-13 spin diffusion experiments, provide enough short, medium, and long-range structural restraints to obtain high-resolution structures of this 2 × 10.4 kDa dimeric protein. Nevertheless, the large number of 13C/15N spins present in this protein, combined with solid-state NMR line widths of about 0.5-1 ppm, induces substantial ambiguities in resonance assignments, preventing 3D structure determination by using distance restraints uniquely assigned on the basis of their chemical shifts. In the second part, we thus demonstrate that an automated iterative assignment algorithm implemented in a dedicated solid-state NMR version of the program ARIA permits to resolve the majority of ambiguities and to calculate a de novo 3D structure from highly ambiguous solid-state NMR data, using a unique fully labeled protein sample. We present, using distance restraints obtained through the iterative assignment process, as well as dihedral angle restraints predicted from chemical shifts, the 3D structure of the fully labeled Crh dimer refined at a root-mean-square deviation of 1.33 Å. IS - 11 JF - J. Am. Chem. Soc. AD - Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS Université Lyon 1, IFR128 BioSciences Lyon-Gerland, 7, passage du Vercors, 69367 Lyon, France, Unité de Bio-Informatique Structurale, URA 2185 CNRS, Institut Pasteur, 25-28 rue du docteur Roux, F-75015 Paris, France, and Department of NMR-based Structural Biology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany EP - 3589 TI - 3D Structure Determination of the Crh Protein from Highly Ambiguous Solid-State NMR Restraints VL - 130 SP - 3579 KW - crh KW - ssnmr AU - Loquet, A AU - Bardiaux, B AU - Gardiennet, C AU - Blanchet, C AU - Baldus, M AU - Nilges, M AU - Malliavin, T AU - Bockmann, A PY - 2008/03/19/ UR - http://dx.doi.org/10.1021/ja078014t ER -