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

摘要

Amide ~(15)N chemical shift anisotropy (CSA) tensors provide quantitative insight into protein structure and dynamics.Experimental determinations of ~(15)N 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 ~(15)N CSA tensor magnitudes in a protein of known structure by three-dimensional MAS solid-state NMR.Isotropic ~(15)N,~(13)C alpha,and ~(13)C' 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 beta1 immunoglobulin binding domain of protein G (GB1) enabled 91 independent determinations of ~(15)N tensors at 51 of the 55 backbone amide sites,for which ~(15)N- ~(13)C alpha and/or ~(15)N-~(13)C' cross-peaks were resolved in the two-dimensional experiment.For 37 ~(15)N signals,both intra- and interresidue correlations were resolved,enabling direct comparison of two experimental data sets to enhance measurement precision.Systematic variations between beta-sheet and alpha-helix residues are observed;the average value for the anisotropy parameter,delta (delta=delta_(zz)-delta_(iso)),for alpha-helical residues is 6 ppm greater than that for the beta-sheet residues.The results show a variation in delta of ~(15)N 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 ~(15)N tensor for K50,a residue that has an atypical,positive value for the backbone phi torsion angle.To our knowledge,this is the most complete experimental analysis of ~(15)N 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 ~(15)N shielding tensor magnitudes.