Rotational diffusion anisotropy of proteins from simultaneous analysis of N-15 and C-13(alpha) nuclear spin relaxation

Current methods of determining the rotational diffusion tensors of proteins in solution by NMR spectroscopy exclusively utilize relaxation rate constants for backbone amide N-15 spins. However, the distributions of orientations of N-H bond vectors are not isotropic in many proteins, and correlations between bond vector orientations reduce the accuracy and precision of rotational diffusion tensors extracted from N-15 spin relaxation data. The inclusion of both C-13(alpha) and N-15 Spin relaxation rate constants increases the robustness of the diffusion tensor analysis because the orientations of the C-alpha-H-alpha bond vectors differ from the orientations of the N-H bond vectors. Theoretical and experimental results for calbindin D-9k, granulocyte colony stimulating factor, and ubiquitin, three proteins with different distributions of N-H and C-alpha-H-alpha bond vectors, are used to illustrate the advantages of the simultaneous utilization of C-13(alpha) and N-15 relaxation data.