NMR Relaxation

Longitudinal and transverse I5N NMR relaxation times in human ubiquitin have been measured at 600MHz 'H frequency with a reproducibility of better than 1%. Two independent measurements of the 15N-{'H} NOE indicate a random error of ca. 0.01, and no values were larger than the theoretical maximum. The relaxation data are incompatible with isotropic rotational diffusion but agree well with an axially symmetric rotational diffusion tensor with a diffusion anisotropy, DldDl, of 1.17. There is no statistically significant further improvement in the fit between the experimental data and those predicted by a fully asymmetric diffusion tensor, confirming that the rotational diffusion tensor of ubiquitin is axially symmetric within experimental uncertainty. The relative ratio of the principal components of the inertia tensor calculated from the X-ray structure is 1.00:0.90:0.64, and the axis with the smallest inertia component makes an angle of 1 1 O with the unique axis of the experimentally determined diffusion tensor. Hydrodynamic calculations agree well with experimental results, provided half a shell of bound water is included and flexibility of the C-terminal residues is accounted for either by omitting them from the calculations or by using conformations for these residues obtained from a Langevin dynamics simulation. The introduction of sensitive indirect detection methods for measuring I5N and 13C NMR relaxation times in has stimulated numerous studies of the rotational diffusion of proteins and the internal dynamics of the backbone. Results are commonly interpreted in terms of a model-free approach' or as values of the spectral density function at various frequencies.*-" In the model-free approach for macromolecules with isotropic overall reorientation, relaxation data are described by a correlation function, C(t) , of the dipolar interaction vector (15N-'H or I3C-IH) given by where S2 is the generalized overall order parameter, z, is the time constant for the isotropic overall motion, and z, is the time constant for the internal motion. When more precise and extensive relaxation data on individual I5N backbone atoms in proteins became available, Clore et a1.I2 noted that for certain residues in a protein, particularly in loop regions, a third time + National Institutes of Health. * Food and Drug Administration. @ Abstract published in Advance ACS Abstracts, December 1, 1995. (1) Kay, L. E.; Jue, T.; Bangerter, B.; Demou, P. C. J. Mugn. Reson. (2) Sklenar, V.; Torchia, D. A.; Bax, A. J. Mugn. Reson. 1987, 73, 375(3)Nirmala, N. R.; Wagner, G. J. Am. Chem. Soc. 1988, 110, 7557(4)Nirmala, N. R.; Wagner, G. J . Mugn. Reson. 1989, 82, 659-661. ( 5 ) Kay, L. E.; Torchia, D. A.; Bax, A. Biochemistry 1989, 28, 8972(6) Palmer, A. G., 111; Skelton, N. J.; Chazin, W. J.; Wright, P. E.; Rance, (7)Lipari, G.; Szabo. A. J. Am. Chem. SOC. 1982, 104, 4546-4558, (8) Peng, J. W.; Wagner, G. J. Magn. Reson. 1992, 98, 308-332. (9) Peng, J. W.; Wagner, G. Biochemistry 1992, 31, 8571-8586. (10) Farrow, N. A.; Zhang, 0.; Szabo, A,; Torchia, D. A,; Kay, L. E. J. (1 1) Ishima, R.; Yamasaki, K.; Saito, M.; Nagayama, K. J . Biomol. NMR 1987, 73, 558-564.