ANISOTROPIC MOLECULAR ROTATIONAL DIFFUSION IN N-15 SPIN RELAXATION STUDIES OF PROTEIN MOBILITY

The backbone dynamics of the uniformly N-15-labeled N-terminal 63-resi due DNA-binding domain of the 434 repressor has been characterized by measurements of the individual N-15 longitudinal relaxation times, T-1 , transverse relaxation times, T-2, and heteronuclear N-15{H}-NOEs at H-1 resonance frequencies of 400 and 750 MHz. The dependence of an app arent spherical top correlation time, tau(R), on the orientation of th e N-H bond vector with respect to the principal axes of the global dif fusion tensor of the protein was used to establish the fact that the d egree of anisotropy of the global molecular tumbling amounts to 1.2, w hich is in good agreement with the values obtained from model calculat ions of the hydrodynamic properties. A model-free analysis showed that even this small anisotropy leads to the implication of artifactual sl ow internal motions for at least two residues when the assumption of i sotropic global motion is used. Additional residues may actually under go internal motions on the same time scale as the global rotational di ffusion, in which case the model-free approach would, however, be inap propriate for quantifying the correlation times and order parameters. Overall, the experiments with 434(1-63) demonstrate that the assumptio n of isotropic rotational reorientation may result in artifacts of mod el-free interpretations of spin relaxation data even for proteins with small deviations from spherical shape.