Anisotropic rotational diffusion of perdeuterated HIV protease from N-15 NMR relaxation measurements at two magnetic

N-15 NMR relaxation times in perdeuterated HIV-1 protease, complexed with the sub-nanomolar inhibitor DMP323, have been measured at 600 and 360 MHz H-1 frequency. The relative magnitudes of the principal components of the inertia tensor, calculated from the X-ray coordinates of the protein-drug complex, are 1.0:0.85:0.44. The relation between the T-1/T-2 ratios observed for the individual backbone amides and their N-H orientation within the 3D structure of the protease dimer yields a rotational diffusion tensor oriented nearly collinear to the inertia tenser. The relative magnitudes of its principal components (1.00:1.11:1.42) are also in good agreement with hydrodynamic modeling results. The orientation and magnitude of the diffusion tensors derived from relaxation data obtained at 360 and 600 MHz are nearly identical. The anisotropic nature of the rotational diffusion has little influence on the order parameters derived from the N-15 T-1 and T-2 relaxation times; however, if anisotropy is ignored, this can result in erroneous identification of either exchange broadening or internal motions on a nanosecond time scale. The average ratio of the T-1 values measured at 360 and 600 MHz is 0.50+/-0.015, which is slightly larger than the value of 0.466 expected for an isotropic rigid rotor with tau(c)=10.7 ns. The average ratio of the T-2 values measured at 360 and 600 MHz is 1.14+/-0.04, which is also slightly larger than the expected ratio of 1.11, This magnetic field dependence of the T-1 and T-2 relaxation times suggests that the spectral density contribution from fast internal motions is not negligible, and that the chemical shift anisotropy of peptide backbone amides, on average, is larger than the 160 ppm value commonly used in N-15 relaxation studies of proteins.