NMR chemical shift tensors (CSTs) in proteins, as well as their orientations, represent an important new restraint class for protein structure refinement and determination. Here, we present the first determination of both CST magnitudes and orientations for 13 Cα and 15 N (peptide backbone) groups in a protein, the β1 IgG binding domain of protein G from Streptococcus spp., GB1. Site-specific 13 Cα and 15 N CSTs were measured using synchronously evolved recoupling experiments in which 13 C and 15 N tensors were projected onto the 1 H-13 C and 1 H-15 N vectors, respectively, and onto the 15 N-13 C vector in the case of 13 Cα. The orientations of the 13 Cα CSTs to the 1 H-13 C and 13 C-15 N vectors agreed well with the results of ab initio calculations, with an rmsd of approximately 8°. In addition, the measured 15 N tensors exhibited larger reduced anisotropies in α-helical versus β-sheet regions, with very limited variation (18 AE 4°) in the orientation of the z-axis of the 15 N CST with respect to the 1 H-15 N vector. Incorporation of the 13 Cα CST restraints into structure calculations, in combination with isotropic chemical shifts, transferred echo double resonance 13 C-15 N distances and vector angle restraints, improved the backbone rmsd to 0.16 Å (PDB ID code 2LGI) and is consistent with existing X-ray structures (0.51 Å agreement with PDB ID code 2QMT). These results demonstrate that chemical shift tensors have considerable utility in protein structure refinement, with the best structures comparable to 1.0-Å crystal structures, based upon empirical metrics such as Ramachandran geometries and χ 1 ∕χ 2 distributions, providing solid-state NMR with a powerful tool for de novo structure determination.magic-angle spinning | dihedral angles | cross validation | nanocrystal | quantum chemistry T he chemical shift is an exquisite and powerful probe of molecular structure, deriving from the interaction of molecular orbitals with an external magnetic field, B 0 . Understanding the relationships between chemical shifts and protein structure has substantial implications for modern nuclear magnetic resonance (NMR) spectroscopy, chemistry, and structural biology (1-12). The chemical shift tensor (CST) is rich with information, even when two-thirds of it is averaged to zero by molecular tumbling in solution or magic-angle spinning (MAS) of solid samples. The remaining isotopic chemical shifts remain an excellent resource for structure determination and validation, and higher-order interactions of the CST have substantial contributions to NMR relaxation (13-19). Therefore, detailed knowledge of CSTs permits a precise analysis of motion (20)(21)(22). Solid-state NMR (SSNMR) of fully aligned samples exploits amide 15 N tensor information to determine the orientations of helices relative to the bilayer (23, 24). We have previously shown that use of a force field in which experimental 13 Cα CSTs are compared with ab initio CSTs [generated as a function of backbone conformation (ϕ, ψ)] significantly improves the precision and...
The study of micro-or nanocrystalline proteins by magic-angle spinning (MAS) solid-state NMR (SSNMR) gives atomic-resolution insight into structure in cases when single crystals cannot be obtained for diffraction studies. Subtle differences in the local chemical environment around the protein, including the characteristics of the co-solvent and the buffer, determine whether a protein will form single crystals. The impact of these small changes in formulation is also evident in the SSNMR spectra, but leads only to correspondingly subtle changes in the spectra. Here we demonstrate that several formulations of GB1 microcrystals yield very high-quality SSNMR spectra, although only a subset of conditions enable growth of single crystals. We have characterized these polymorphs by X-ray powder diffraction and assigned the SSNMR spectra. Assignments of the 13 C and 15 N SSNMR chemical shifts confirm that the backbone structure is conserved, indicative of a common protein fold, but sidechain chemical shifts are changed on the surface of the protein, in a manner dependent upon crystal packing and electrostatic interactions with salt in the mother liquor. Our results demonstrate the ability of SSNMR to reveal minor structural differences among crystal polymorphs. This ability has potential practical utility for studying formulation chemistry of industrial and therapeutic proteins, as well as for deriving fundamental insights into the phenomenon of single crystal growth.
Solid-state NMR has emerged as an important tool for structural biology and chemistry, capable of solving atomic-resolution structures for proteins in membrane-bound and aggregated states. Proton detection methods have been recently realized under fast magic-angle spinning conditions, providing large sensitivity enhancements for efficient examination of uniformly labeled proteins. The first and often most challenging step of protein structure determination by NMR is the site-specific resonance assignment. Here we demonstrate resonance assignments based on high-sensitivity proton-detected three-dimensional experiments for samples of different physical states, including a fully-protonated small protein (GB1, 6 kDa), a deuterated microcrystalline protein (DsbA, 21 kDa), a membrane protein (DsbB, 20 kDa) prepared in a lipid environment, and the extended core of a fibrillar protein (α-synuclein, 14 kDa). In our implementation of these experiments, including CONH, CO(CA)NH, CANH, CA(CO)NH, CBCANH, and CBCA(CO)NH, dipolar-based polarization transfer methods have been chosen for optimal efficiency for relatively high protonation levels (full protonation or 100 % amide proton), fast magic-angle spinning conditions (40 kHz) and moderate proton decoupling power levels. Each H–N pair correlates exclusively to either intra- or inter-residue carbons, but not both, to maximize spectral resolution. Experiment time can be reduced by at least a factor of 10 by using proton detection in comparison to carbon detection. These high-sensitivity experiments are especially important for membrane proteins, which often have rather low expression yield. Proton-detection based experiments are expected to play an important role in accelerating protein structure elucidation by solid-state NMR with the improved sensitivity and resolution.
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