Direct Determination of Protein Rotational Diffusion Tensors and Generalized Order Parameters from Multifield 15N NMR Spin Relaxation.

NMR relaxation of backbone amide 15N spins enables characterization of overall and intramolecular dynamic properties of proteins, including overall rotational diffusion on nanosecond time scales; internal conformational dynamics on picosecond-nanosecond time scales, through generalized order parameters and effective correlation times; and slow inter- and intramolecular processes on microsecond-millisecond time scales, termed chemical exchange. Selection between competing models for dynamic processes, such as various versions of the model-free formalism, complicates applications of these otherwise powerful methods. Chemical exchange systematically increases the transverse relaxation rate constant R2 and is a strong confounding factor in model-free or other interpretations of 15N spin relaxation measurements. The present work presents robust approaches, using 15N spin relaxation data acquired at three or more static magnetic fields, to detect and remove chemical exchange contributions to R2; to determine the local isotropic rotational correlation time and generalized order parameter for individual 15N sites without model selection; and to recursively maximize the number of local isotropic rotational correlation times used to determine the overall rotational diffusion tensor. These approaches are applied to 15N relaxation data acquired at 500, 700, and 900 MHz for Escherichia coli ribonuclease HI, and compared to conventional analyses using the model-free formalism. These methods enable facile determination of the most important parameters describing protein dynamics: the rotational diffusion tensor, generalized order parameters, and chemical exchange constants, and broaden the scope of NMR spin relaxation methods.