Restraints on backbone conformations in solid state NMR studies of uniformly labeled proteins from quantitative amide ¹⁵N-¹⁵N and carbonyl ¹³C-¹³C dipolar recoupling data

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• 作者：Kan-Nian Hu^a ; ¹ ; Wei Qiang^a ; Guillermo A. Bermejo^b ; Charles D. Schwieters^b ; Robert Tycko^a ; ^{rtycko@comcast.net} ; ^{robertty@mail.nih.gov}
• 关键词：Magic-angle spinning ; Protein structure ; Pulse sequences
• 刊名：Journal of Magnetic Resonance
• 出版年：2012
• 期刊代码：126_10907807
• 类别：ch
• 出版时间：May, 2012
• 卷：218
• 期：Complete
• 页码：115-127
• 文件大小：2742 K

摘要

Recent structural studies of uniformly ¹⁵N, ¹³C-labeled proteins by solid state nuclear magnetic resonance (NMR) rely principally on two sources of structural restraints: (i) restraints on backbone conformation from isotropic ¹⁵N and ¹³C chemical shifts, based on empirical correlations between chemical shifts and backbone torsion angles; (ii) restraints on inter-residue proximities from qualitative measurements of internuclear dipole-dipole couplings, detected as the presence or absence of inter-residue crosspeaks in multidimensional spectra. We show that site-specific dipole-dipole couplings among ¹⁵N-labeled backbone amide sites and among ¹³C-labeled backbone carbonyl sites can be measured quantitatively in uniformly-labeled proteins, using dipolar recoupling techniques that we call ¹⁵N-BARE and ¹³C-BARE (BAckbone REcoupling), and that the resulting data represent a new source of restraints on backbone conformation. ¹⁵N-BARE and ¹³C-BARE data can be incorporated into structural modeling calculations as potential energy surfaces, which are derived from comparisons between experimental ¹⁵N and ¹³C signal decay curves, extracted from crosspeak intensities in series of two-dimensional spectra, with numerical simulations of the ¹⁵N-BARE and ¹³C-BARE measurements. We demonstrate this approach through experiments on microcrystalline, uniformly ¹⁵N, ¹³C-labeled protein GB1. Results for GB1 show that ¹⁵N-BARE and ¹³C-BARE restraints are complementary to restraints from chemical shifts and inter-residue crosspeaks, improving both the precision and the accuracy of calculated structures.