Thursday, May 18, 2006

JPCB, Rienstra, 15N CSA in proteins; 3D MAS NMR

J. Phys. Chem. B, ASAP Article 10.1021/jp060507h S1520-6106(06)00507-4

Determinations of 15N Chemical Shift Anisotropy Magnitudes in a Uniformly 15N,13C-Labeled Microcrystalline Protein by Three-Dimensional Magic-Angle Spinning Nuclear Magnetic Resonance Spectroscopy

Benjamin J. Wylie, W. Trent Franks, and Chad M. Rienstra*

Department of Chemistry, Department of Biochemistry, and Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801


Amide 15N chemical shift anisotropy (CSA) tensors provide quantitative insight into protein structure and dynamics. Experimental determinations of 15N CSA tensors in biologically relevant molecules have typically been performed by NMR relaxation studies in solution, goniometric analysis of single-crystal spectra, or slow magic-angle spinning (MAS) NMR experiments of microcrystalline samples. Here we present measurements of 15N CSA tensor magnitudes in a protein of known structure by three-dimensional MAS solid-state NMR. Isotropic 15N, 13C, and 13C' chemical shifts in two dimensions resolve site-specific backbone amide recoupled CSA line shapes in the third dimension. Application of the experiments to the 56-residue 1 immunoglobulin binding domain of protein G (GB1) enabled 91 independent determinations of 15N tensors at 51 of the 55 backbone amide sites, for which 15N-13C and/or 15N-13C' cross-peaks were resolved in the two-dimensional experiment. For 37 15N signals, both intra- and interresidue correlations were resolved, enabling direct comparison of two experimental data sets to enhance measurement precision. Systematic variations between -sheet and -helix residues are observed; the average value for the anisotropy parameter, ( = zz - iso), for -helical residues is 6 ppm greater than that for the -sheet residues. The results show a variation in of 15N amide backbone sites between -77 and -115 ppm, with an average value of -103.5 ppm. Some sites (e.g., G41) display smaller anisotropy due to backbone dynamics. In contrast, we observe an unusually large 15N tensor for K50, a residue that has an atypical, positive value for the backbone torsion angle. To our knowledge, this is the most complete experimental analysis of 15N CSA magnitude to date in a solid protein. The availability of previous high-resolution crystal and solution NMR structures, as well as detailed solid-state NMR studies, will enhance the value of these measurements as a benchmark for the development of ab initio calculations of amide 15N shielding tensor magnitudes.

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