Ultrahigh resolution protein structures using NMR chemical shift tensors

Abstract

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...

Recoupling of chemical shift anisotropy by R-symmetry sequences in magic angle spinning NMR spectroscopy

“…Therefore, the latter family of sequences is applicable for measurements of CSA parameters in systems with strong homonuclear dipolar couplings, such as uniformly-13 C labeled biological solids. We demonstrate RNCSA NMR experiments and numerical simulations establishing the utility of this approach to the measurements of 13 …”

“…In these recoupling techniques, the required rf field strengths are generally so high that, in practice, these sequences are only applicable under low or moderate MAS conditions. In addition, since homonuclear dipoledipole interactions cannot be suppressed efficiently, most of the existing recoupling methods are not suitable for CSA measurements in strongly coupled systems, i.e., uniformly 13 C enriched samples. In this work, we demonstrate that R-type symmetry pulse sequences [33][34][35][36] are well suited for recoupling of CSA interactions under MAS.…”

“…In contrast, the γ -encoded symmetry sequences for σ 2 -CSA recoupling have smaller scaling factors but they suppress efficiently the homonuclear dipole-dipole interactions. Therefore, the latter family of sequences is well suited for the measurements of CSA parameters in systems with strong homonuclear dipolar couplings, such as uniformly- 13 C labeled biological solids. We present RNCSA experiments and simulations establishing the utility of this approach to the measurements of 13 15 N]-Ala and natural abundance L-alanine were purchased from Cambridge Isotope Laboratories.…”

“…[1][2][3][4][5][6][7][8][9][10][11][12][13] Even though select non-isotropic properties of chemical shift tensors can be obtained through solution-state NMR approaches, solid-state NMR spectroscopy is far more suitable for deriving the anisotropic information. 14 In solids, CSA can be directly measured by recording powder patterns in static powder samples, but these experiments suffer from poor sensitivity, spectral overlap when multiple chemical sites are present, and from broadening and distortion to the CSA line shapes introduced by a) Author to whom correspondence should be addressed.…”

Zero-quantum stochastic dipolar recoupling in solid state nuclear magnetic resonance

“…6 shows examples of 2D 13 C-13 C NMR spectra of protein GB1, a 56-residue model protein 37 that has been the subject of many previous solid state NMR studies, [42][43][44][45][46][47] obtained with the pulse sequence in Fig. 2(c).…”