Protein Structure Determination by High-Resolution Solid-State NMR Spectroscopy: Application to Microcrystalline Ubiquitin Stephan G. Zech,* ,²,§ A. Joshua Wand, ‡ and Ann E. McDermott* ,² 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 Received January 17, 2005; E-mail: Stephan.Zech@web.de; aem5@columbia.edu. 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 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. 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. Introduction Over the past few years, there have been remarkable advances in solid-state NMR (ssNMR) experiments for characterization of protein structure and function. In a wide variety of systems the protein’s insolubility made X-ray crystallography or solution NMR unsuitable, while questions on structure and dynamics can be addressed with ssNMR. 1-5 These efforts typically involve structurally homogeneous samples and utilize recently developed pulse sequences for sequential correlation of resonances, detec- tion of tertiary contacts, and characterization of torsion angles. Excellent NMR line widths can be achieved for microcrys- talline or precipitated hydrated globular systems using magic angle spinning (MAS) methods. More recent advances in high- field instrumentation and pulse sequences for chemical shift correlation experiments led to more efficient methods of assigning solid-state proteins with extensive isotopic enrichment and a rapid succession of studies on small globular proteins has become evident, including BPTI, 6 the R-spectrin SH3 domain, 7,8,9 the catabolite repression phosphocarrier protein (Crh), 10 human ubiquitin, 11,12 thioredoxin, 13 the immunoglobulin binding domain 1 of streptococcal protein G (GB1), 14 and peptides such as neurotensin, 15 mastoparan-X, 16 and a fibrillar peptide fragment of transthyretin. 17 For tertiary structure determination, or fold definition, long- range constraints have typically served a critical role. It has been recently demonstrated that simple spin diffusion experiments, if combined with strategic labeling schemes, are sufficient to determine a moderate resolution structure of a protein. 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