Determinations of15N chemical shift anisotropy magnitudes in a uniformly15N,13C-labeled microcrystalline protein by three-dimensional magic-angle spinning nuclear magnetic resonance spectroscopy

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

Research output: Contribution to journalArticlepeer-review

59 Scopus citations

Abstract

Amide 15N chemical shift anisotropy (CSA) tensors provide quantitative insight into prqtein 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. Isotropie 15N, 13Ca, 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 β1 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-13Cα 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 β-sheet and α-helix residues are observed; the average value for the anisotropy parameter, δ (δ = δzz - δiso), for α-helical residues is 6 ppm greater than that for the β-sheet residues. The results show a variation in δ 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 φ 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.

Original languageEnglish
Pages (from-to)10926-10936
Number of pages11
JournalJournal of Physical Chemistry B
Volume110
Issue number22
DOIs
StatePublished - Jun 8 2006

Fingerprint Dive into the research topics of 'Determinations of<sup>15</sup>N chemical shift anisotropy magnitudes in a uniformly<sup>15</sup>N,<sup>13</sup>C-labeled microcrystalline protein by three-dimensional magic-angle spinning nuclear magnetic resonance spectroscopy'. Together they form a unique fingerprint.

Cite this