Density functional calculations of chemical shielding of backbone 15N in helical residues of protein G.
ABSTRACT We performed density functional calculations of backbone (15)N chemical shielding tensors in selected helical residues of protein G. Here we describe a computationally efficient methodology to include most of the important effects in the calculation of chemical shieldings of backbone (15)N. We analyzed the role of long-range intra-protein electrostatic interactions by comparing models with different complexity in vacuum and in charge field. Our results show that the dipole moment of the alpha-helix can cause significant deshielding of (15)N; therefore, it needs to be considered when calculating (15)N chemical shielding. We found that it is important to include interactions with the side chains that are close in space when the charged form for ionizable side chains is adopted in the calculation. We also illustrate how the ionization state of these side chains can affect the chemical shielding tensor elements. Chemical shielding calculations using a 8-residue fragment model in vacuum and adopting the charged form of ionizable side chains yield a generally good agreement with experimental data.
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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 ± 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.Proceedings of the National Academy of Sciences 10/2011; 108(41):16974-9. · 9.68 Impact Factor