Evaluation of two simplified 15N-NMR methods for determining micros-ms dynamics of proteins

Two methods for estimating the microsecond-millisecond dynamics in proteins from only two 15N relaxation parameters at one magnetic field strength are investigated. Thus, the chemical exchange contribution, R(ex), to the transversal relaxation rate, which contains the dynamics information, is evaluated by two methods: (i) one in which the R(ex) term is derived from the 15N R1 and R2 relaxation rates alone, and (ii) one in which it is obtained from the transversal dipole-chemical shift anisotropy (CSA) cross-correlation rate, eta(xy), and the R2 rate. Since the R1, R2, and eta(xy) experiments are fast and sensitive, both methods are attractive in studies where large amounts of dynamical information are required. However, both methods are liable to effects that can compromise the estimation of the R(ex) terms. In the R2/R1 method, internal ps-ns dynamics and rotational anisotropy can interfere with the determination of R(ex), while in the R2/eta(xy) method it can be affected by variations in the 15N chemical shift anisotropy. Here, the applicability of the two methods is investigated using plastocyanin from Anabaena variabilis as an example, and the quality of the obtained R(ex) terms is evaluated both theoretically and experimentally. It is found that the R2/R1 method gives reliable R(ex) terms if the protein is relatively rigid and tumbles fast and nearly isotropically in solution, as for instance plastocyanin, and is preferable in such cases. In contrast, the R2/eta(xy) method gives better results if the protein is flexible or highly non-spherical and can be used for such proteins, if the sequential variation in the 15N chemical shift anisotropy is negligible. For exchange terms <1 s(-1) neither method is reliable.