Protein Structure Determination by High-Resolution Solid-State NMR Spectroscopy: Application to Microcrystalline Ubiquitin
Stephan G. Zech,* A. Joshua Wand, and Ann E. McDermott*
Contribution from the Department of Chemistry, Columbia University, 3000 Broadway Mail Code 3113, New York, New York 10027, and Department of Biochemistry and Biophysics, University of Pennsylvania, The Johnson Research Foundation, Philadelphia, Pennsylvania 19104
J. Am. Chem. Soc., 127 (24), 8618 -8626, 2005.
Abstract:
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 13C-13C correlation spectroscopy under magic angle spinning conditions. High-resolution 13C spectra have been acquired from hydrated microcrystals of site-directed 13C-enriched ubiquitin. Interresidue 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 Å. 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.