The understanding of protein function is incomplete without the study of protein dynamics. NMR spectroscopy is valuable for probing nanosecond and picosecond dynamics via relaxation studies. The use of 15 N relaxation to study backbone dynamics has become virtually standard. Here, we propose to measure the relaxation of additional nuclei on each peptide plane allowing for the observation of anisotropic local motions. This allows the nature of local motions to be characterized in proteins. As an example, semilocal rotational motion was detected for part of a helix of the protein Escherichia coli f lavodoxin.The functions of proteins are dictated by their threedimensional structures as well as by their dynamic behavior. In many cases, dynamics such as flap and domain movements as well as overall conformational changes are essential for the full and correct function of proteins (1-4).One way to measure the dynamic behavior of proteins on the nanosecond and picosecond time scale is via NMR relaxation (5-7). Relaxation is caused by fluctuations of interaction energies, e.g., dipole-dipole energies, as the internuclear interaction vectors are reoriented by thermal motion. For example, the time constant for longitudinal dipolar relaxation of spin I by spin S, T 1 , is given bywhere ␥ I and ␥ S are the gyromagnetic ratios of I and S, r IS is the internuclear distance between I and S, and ប ϭ Planck's constant͞2. The spectral density functions, J(), which represent the intensity of the internuclear vector motion at the NMR frequencies (typically 10 9 rad͞s), are the actual reporters of the reorientational dynamics of the internuclear interaction vectors (8, 9).Relaxation measurements are typically carried out with 15 N-labeled proteins (10 -11). By using two-or threedimensional NMR techniques, the values for the J() can be obtained for virtually all N-NH vectors in the molecule. Using the model-free approach (12, 13), one obtains from J() the overall tumbling with characteristic correlation time c and the local mobility characterized by the correlation time e , on a residue specific basis, according towith M ϭ c e c ϩ e .[2]The order parameter, S 2 , describes the amount of local mobility, with S 2 ϭ 1 for no local motion and S 2 ϭ 0 for completely unrestricted local motion of the NH vectors. For a typical relaxation study, S 2 , c , and e are reported for the different NH sites. This is a powerful method that has allowed the detection of extensive local dynamics, especially of loops and termini, in many proteins. A general shortcoming, however, is that these existing methods cannot identify what the local motions are. This seriously hampers our understanding of the biological significance of the local motions.We propose here that to better obtain insight into the nature of the local motion, one must measure, at any given location, the relaxation of two or more interactions corresponding to internuclear vectors that lie at some fixed angle to each other. Thus, the reorientation of each of these vectors is charact...