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

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.