Assessing potential bias in the determination of rotational correlation times of proteins by NMR relaxation

The various factors that influence the reliable and efficient determination of the correlation time describing molecular reorientation of proteins by NMR relaxation methods are examined, Nuclear Overhauser effects, spin-lattice, and spin-spin relaxation parameters of N-15 NMR relaxation in ubiquitin have been determined at 17.6, 14.1, 11.7 and 9.3 Tesla. This unusually broad set of relaxation parameters has allowed the examination of the influence of chemical shift anisotropy, the functional form of the model-free spectral density, and the reliability of determined spin-spin relaxation parameters on the characterization of global tumbling of the protein. Treating the N-15 chemical shift anisotropy (CSA) as an adjustable parameter, a consensus value of -170 +/- 15 ppm for the breadth of the chemical shift tensor and a global isotropic correlation time of 4.1 ns are found when using the model-free spectral density to fit T-1 and NOE data from all fields. The inclusion of T-2 relaxation parameters in the determination of the global correlation time results in its increase to 4.6 ns. This apparent inconsistency may explain a large portion of the discrepancy often found between NMR- and fluorescence-derived tau(m) values for proteins. The near identity of observed T-2 and T-1 rho values suggests that contributions from slow motions are not the origin of the apparent inconsistency with obtained T-1 and NOE data. Various considerations suggest that the origin of this apparent discrepancy may reside in a contribution to the spectral density at zero frequency that is not represented by the simple model-free formalism in addition to the usual experimental difficulties associated with the measurement of these relaxation parameters. Finally, an axially symmetric diffusion tensor for ubiquitin is obtained using exclusively T-1 and NOE data. A recommendation is reached on the types and combinations of relaxation data that can be used to reliably determine tau(m) values. It is also noted that the reliable determination of tau(m) values from N-15 T-1 and NOE relaxation parameters will become increasingly difficult as tau(m) increases.