Magic-Angle Spinning Solid-State NMR Spectroscopy of the 1 Immunoglobulin Binding Domain of Protein G (GB1): 15 N and 13 C Chemical Shift Assignments and Conformational Analysis W. Trent Franks, Donghua H. Zhou, Benjamin J. Wylie, Brian G. Money, Daniel T. Graesser, Heather L. Frericks, Gurmukh Sahota, and Chad M. Rienstra* Contribution from the Department of Chemistry, UniVersity of Illinois at Urbana-Champaign, 600 South Mathews AVenue, Urbana, Illinois 61801 Received September 10, 2004; E-mail: rienstra@scs.uiuc.edu Abstract: Magic-angle spinning solid-state NMR (SSNMR) studies of the 1 immunoglobulin binding domain of protein G (GB1) are presented. Chemical shift correlation spectra at 11.7 T (500 MHz 1 H frequency) were employed to identify signals specific to each amino acid residue type and to establish backbone connectivities. High sensitivity and resolution facilitated the detection and assignment of every 15 N and 13 C site, including the N-terminal (M1) 15 NH3, the C-terminal (E56) 13 C′, and side-chain resonances from residues exhibiting fast-limit conformational exchange near room temperature. The assigned spectra lend novel insight into the structure and dynamics of microcrystalline GB1. Secondary isotropic chemical shifts report on conformation, enabling a detailed comparison of the microcrystalline state with the conformation of single crystals and the protein in solution; the consistency of backbone conformation in these three preparations is the best among proteins studied so far. Signal intensities and line widths vary as a function of amino acid position and temperature. High-resolution spectra are observed near room temperature (280 K) and at <180 K, whereas resolution and sensitivity greatly degrade substantially near 210 K; the magnitude of this effect is greatest among the side chains of residues at the intermolecular interface of the microcrystal lattice, which we attribute to intermediate-rate translational diffusion of solvent molecules near the glass transition. These features of GB1 will enable its use as an excellent model protein not only for SSNMR methods development but also for fundamental studies of protein thermodynamics in the solid state. Introduction Robust and efficient methods for determining atomic resolu- tion structures of macroscopically disordered proteins are highly sought after, due to the importance of membrane proteins as pharmaceutical targets 1 and the roles that insoluble aggregates of peptides (fibrils) play in neurodegenerative diseases. 2 Beyond the direct implications of atomic resolution data for rational drug design, experimental measurements of structural and dynamic parameters in the solid state enhance the fundamental under- standing of protein thermodynamics and provide valuable benchmarks for comparison to theoretical models of protein folding, electrostatics, and dynamics. It is well-known that solid- state NMR (SSNMR) can directly probe anisotropic parameters (such as chemical shift anisotropies and dipolar couplings) of interest to these problems. 3,4 However, the rate at which such data have been extracted from SSNMR spectra has historically been inadequate for site-specific measurements to be made throughout entire proteins; hence systematic comparisons of experiment versus theory have rarely been possible. More efficient methods to acquire and assign SSNMR spectra of entire proteins will permit such analyses and assist in developing a more complete understanding of the differences that sometimes exist between X-ray diffraction and solution NMR structures of proteins. 5 Thus, in recent years major research efforts have been aimed at developing methods to accelerate the rate of data accumulation and interpretation by SSNMR, principally by studying uniformly- 13 C, 15 N-enriched peptides and proteins. In particular, magic- angle spinning (MAS) SSNMR methodology for de novo assignment and structure determination of uniformly- 13 C, 15 N- labeled peptides and proteins has rapidly advanced. Landmark high-field (750-800 MHz) MAS studies demonstrated that 2D 13 C- 13 C correlation spectra of basic pancreatic trypsin inhibitor (BPTI) 6 and a 62-residue R-spectrin SH3 domain 7 could be acquired with sub-ppm resolution. Site-resolved signals from these spectra could be identified by residue type and assigned by comparison to solution NMR data. Subsequently, 2D 15 N- 13 C spectra established sequence-specific correlations, critical (1) Leibmann, C. Curr. Pharm. Design 2004, 10, 1937-1958. (2) Caughey, B.; Lansbury, P. T. Annu. ReV. Neurosci. 2003, 26, 267-298. (3) McDowell, L. M.; Schaefer, J. Curr. Opin. Struct. Biol. 1996, 6, 624- 629. (4) Tycko, R. Prog. Nucl. Magn. Reson. Spectrosc. 2003, 42, 53-68. (5) Smith, S. O.; Farr-Jones, S.; Griffin, R. G.; Bachovchin, W. W. Science 1989, 244, 961-964. (6) McDermott, A.; Polenova, T.; Bockmann, A.; Zilm, K. W.; Paulsen, E. K.; Martin, R. W.; Montelione, G. T. J. Biomol. NMR 2000, 16, 209-219. (7) Pauli, J.; van Rossum, B.; Forster, H.; de Groot, H. J. M.; Oschkinat, H. J. Magn. Reson. 2000, 143, 411-416. Published on Web 08/11/2005 10.1021/ja044497e CCC: $30.25 © 2005 American Chemical Society J. AM. CHEM. SOC. 2005, 127, 12291-12305 9 12291