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
May 2012
Publication year: 2012
Source:Journal of Magnetic Resonance, Volume 218
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.
Graphical abstract
Highlights
? 15N and 13C backbone recoupling (BARE) yields accurate dipolar dephasing curves. ? Torsion angle restraints detected through 2D NCACX, NCOCX, or COCA crosspeaks. ? 15N- and 13C-BARE restraints incorporated as 3D potential surfaces in Xplor-NIH. ? 15N-BARE and 13C-BARE restraints improve structural precision and accuracy.
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