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

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 = δ_zz - δ_iso), 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.