Assigning large proteins in the solid state: a MAS NMR resonance assignment strategy using selectively and extensively 13C-labelled proteins Export

@article{citeulike:5215845, abstract = {Abstract\ \ In recent years, solid-state magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) has been growing into an important technique to study the structure of membrane proteins, amyloid fibrils and other protein preparations which do not form crystals or are insoluble. Currently, a key bottleneck is the assignment process due to the absence of the resolving power of proton chemical shifts. Particularly for large proteins (approximately \>150 residues) it is difficult to obtain a full set of resonance assignments. In order to address this problem, we present an assignment method based upon samples prepared using [1,3-13C]- and [2-13C]-glycerol as the sole carbon source in the bacterial growth medium (so-called selectively and extensively labelled protein). Such samples give rise to higher quality spectra than uniformly [13C]-labelled protein samples, and have previously been used to obtain long-range restraints for use in structure calculations. Our method exploits the characteristic cross-peak patterns observed for the different amino acid types in 13C-13C correlation and 3D NCACX and NCOCX spectra. An in-depth analysis of the patterns and how they can be used to aid assignment is presented, using spectra of the chicken α-spectrin SH3 domain (62 residues), αB-crystallin (175 residues) and outer membrane protein G (OmpG, 281 residues) as examples. Using this procedure, over 90\% of the Cα, Cβ, C′ and N resonances in the core domain of αB-crystallin and around 73\% in the flanking domains could be assigned (excluding 24 residues at the extreme termini of the protein).}, author = {Higman, Victoria and Flinders, Jeremy and Hiller, Matthias and Jehle, Stefan and Markovic, Stefan and Fiedler, Sebastian and van Rossum, Barth-Jan and Oschkinat, Hartmut}, citeulike-article-id = {5215845}, citeulike-linkout-0 = {http://dx.doi.org/10.1007/s10858-009-9338-7}, citeulike-linkout-1 = {http://www.springerlink.com/content/p6pn734835220g1g}, day = {1}, doi = {10.1007/s10858-009-9338-7}, journal = {Journal of Biomolecular NMR}, keywords = {13c, assignments, ssnmr}, month = {August}, number = {4}, pages = {245--260}, posted-at = {2009-07-21 08:21:07}, priority = {2}, title = {Assigning large proteins in the solid state: a MAS NMR resonance assignment strategy using selectively and extensively 13C-labelled proteins}, url = {http://dx.doi.org/10.1007/s10858-009-9338-7}, volume = {44}, year = {2009} }

TY - JOUR ID - citeulike:5215845 L3 - citeulike-article-id:5215845 N2 - Abstract  In recent years, solid-state magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) has been growing into an important technique to study the structure of membrane proteins, amyloid fibrils and other protein preparations which do not form crystals or are insoluble. Currently, a key bottleneck is the assignment process due to the absence of the resolving power of proton chemical shifts. Particularly for large proteins (approximately >150 residues) it is difficult to obtain a full set of resonance assignments. In order to address this problem, we present an assignment method based upon samples prepared using [1,3-13C]- and [2-13C]-glycerol as the sole carbon source in the bacterial growth medium (so-called selectively and extensively labelled protein). Such samples give rise to higher quality spectra than uniformly [13C]-labelled protein samples, and have previously been used to obtain long-range restraints for use in structure calculations. Our method exploits the characteristic cross-peak patterns observed for the different amino acid types in 13C-13C correlation and 3D NCACX and NCOCX spectra. An in-depth analysis of the patterns and how they can be used to aid assignment is presented, using spectra of the chicken α-spectrin SH3 domain (62 residues), αB-crystallin (175 residues) and outer membrane protein G (OmpG, 281 residues) as examples. Using this procedure, over 90% of the Cα, Cβ, C′ and N resonances in the core domain of αB-crystallin and around 73% in the flanking domains could be assigned (excluding 24 residues at the extreme termini of the protein). IS - 4 JF - Journal of Biomolecular NMR EP - 260 TI - Assigning large proteins in the solid state: a MAS NMR resonance assignment strategy using selectively and extensively 13C-labelled proteins VL - 44 SP - 245 KW - 13c KW - assignments KW - ssnmr AU - Higman, Victoria AU - Flinders, Jeremy AU - Hiller, Matthias AU - Jehle, Stefan AU - Markovic, Stefan AU - Fiedler, Sebastian AU - van Rossum, Barth-Jan AU - Oschkinat, Hartmut PY - 2009/08/01/ UR - http://dx.doi.org/10.1007/s10858-009-9338-7 ER -