De Novo Determination of Bond Orientations and Order Parameters from Residual Dipolar Couplings with High Accuracy

Abstract

We report the de novo determination of ¹⁵N−¹H bond orientations and motional order parameters for the protein ubiquitin with high accuracy based solely on NMR residual dipolar coupling measurements made in six distinct alignment media. The resulting bond orientations are in agreement with RDC-refined orientations of either solid or solution state coordinates to within approximately 2°, which is also the estimated precision of the resulting orientations. The squared generalized order parameters, which reflect amplitudes of motion spanning the picosecond to millisecond time scales, exhibit a correlation with picosecond time scale order parameters derived from conventional NMR ¹⁵N spin relaxation methods. Provided that RDC measurements can be obtained using many different alignment media, this approach (called direct interpretation of dipolar couplings) may significantly impact the attainable accuracy and the molecular weight range accessible to NMR structure determination in the solution state, as well as provide a route for the study of motions occurring on the nanosecond to microsecond time scales, which have been traditionally difficult to study at atomic resolution.