Protein Conformational Flexibility from Structure-Free Analysis of NMR Dipolar Couplings: Quantitative and Absolute Determination of Backbone Motion in Ubiquitin

摘要

Molecular dynamics play an essential role in controlling the biological activity of proteins. NMR residual dipolar couplings (RDCs) are uniquely sensitive to conformational detail and thus offer a very attractive approach to the characterization of protein dynamics on all time scales up to the millisecond. The simple averaging properties of RDCs make them amenable to rigorous interpretation in terms of protein structure and dynamics. Assumptions made when analyzing protein dynamics from RDCs, as well as the robustness of the resulting dynamic description, can be largely substantiated through self-consistency checks. The recognition of these clear advantages has led to the development of several approaches to the characterization of protein-backbone motions from RDCs, including analytical deconvo-lution of the amplitudes and anisotropies of bond vectors or structural motifs, ensemble-averaging by restrained-molecular-dynamics simulation,and direct comparison to unrestrained molecular dynamics (MD).In previous studies, however, only relative motional amplitudes could be determined directly from RDCs, because internal motional amplitudes and alignment strength cannot be separated easily. To derive absolute motional amplitudes from RDCs, NMR-relaxation-derived S~2_(Rel) order parameters have been used as upper limits of the corresponding RDC-derived order param-eters S~2_(RDC).