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

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