Protein structure determination by high-resolution solid-state NMR spectroscopy: application to microcrystalline ubiquitin.
High-resolution solid-state NMR spectroscopy has become a promising method for the determination of three-dimensional protein structures for systems which are difficult to crystallize or exhibit low solubility. Here we describe the structure determination of microcrystalline ubiquitin using 2D (13)C-(13)C correlation spectroscopy under magic angle spinning conditions. High-resolution (13)C spectra have been acquired from hydrated microcrystals of site-directed (13)C-enriched ubiquitin. Inter-residue carbon-carbon distance constraints defining the global protein structure have been evaluated from 'dipolar-assisted rotational resonance' experiments recorded at various mixing times. Additional constraints on the backbone torsion angles have been derived from chemical shift analysis. Using both distance and dihedral angle constraints, the structure of microcrystalline ubiquitin has been refined to a root-mean-square deviation of about 1 A. The structure determination strategies for solid samples described herein are likely to be generally applicable to many proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy.
Zech
Stephan G
SG
Department of Chemistry, Columbia University, 3000 Broadway Mail Code 3113, New York, New York 10027, USA. Stephan.Zech@web.de
Wand
A Joshua
AJ
McDermott
Ann E
AE
D002247:Carbon Isotopes; D003460:Crystallization; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D025801:Ubiquitin / Q000737:chemistry*
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D025801:Ubiquitin