Refinement of the protein backbone angle psi in NMR structure calculations

Cross-correlated relaxation rates involving the C-alpha-H-alpha dipolar int eraction and the carbonyl (C') chemical shift anisotropy (CSA) have been me asured using two complementary 3D experiments. We show that the protein bac kbone angle psi can be directly refined against such cross-correlated relax ation rates (Gamma(H alpha C alpha,C')) and the three-bond H/D isotope effe ct on the C-alpha chemical shifts ((3)Delta C-(ND)(alpha)). By simultaneous ly using both experimental parameters as restraints during NMR structure ca lculations, a unique value for the backbone angle psi is defined. We have a pplied the new refinement method to the alpha-Spectrin SH3 domain (a beta-s heet protein) and to the Sgs1p HRDC domain (an alpha-helical protein) and s how that the quality of the NMR structures is substantially improved, judgi ng from the atomic coordinate precision and the Ramachandran map. In additi on, the psi-refined NMR structures of the SH3 domain deviate less from the 1.8 Angstrom crystal structure, suggesting an improved accuracy. The propos ed refinement method can be used to significantly improve the quality of NM R structures and will be applicable to larger proteins.