To investigate the backbone dynamics of roteins I5N longitudinal and transverse relaxation experiments combined with (lH, r5N] NOE measurements together with molecular dynamics simulations were carried out using ribonuclease TI and the complex of ribonuclease TI with 2'GMP as a model protein. The intensity decay of individual amide cross peaks in a series of ('H, "N)HsQC spectra with appropriate relaxation pyiods was fitted to a single exponential by using a simplex algorithm in order to obtain and T, relaxation times. The relaxation times were analyzed in terms of the "model-free" approach introduced by Lipari and Szabo. In addition, a nanosecond molecular dynamics (MD) simulation of ribonuclease T , and its 2'GMP complex in water was carried out. The angular reorientations of the backbone amide groups were classified with several coordinate frames following a transformation of NH vector trajectories. In this study, NH librations and backbone dihedral angle fluctuations were distinguished. The NH bond librations were found to be similar for all amides as characterized by correlation times of librational motions in a subpicosecond scale. The angular amplitudes of these motions were found to be about 10"-12" for out-of-plane displacements and 3"-5" for the in-plane displacement. The contributions from the much slower backbone dihedral angle fluctuations strongly depend on the secondary structure. The dependence of the amplitude of local motion on the residue location in the backbone is in good agreement with the results of NMR relaxation measurements and the X-ray data. The protein dynamics is characterized by a highly restricted local motion of those parts of the backbone with defined secondary structure as well as by a high flexibility in loop regions. Comparison of the MD and NMR data of the free liganded enzyme ribonuclease TI clearly indicates a restriction of the mobility within certain regions of the backbone upon inhibitor binding.