Backbone dynamics of barstar: A < sup > 15 < /sup > N NMR relaxation study

Abstract

Backbone dynamics of uniformly 15N-labeled barstar have been studied at 32°C, pH 6.7, by using 15N relaxation data obtained from proton-detected 2D {1H}-15N NMR spectroscopy. 15N spin-lattice relaxation rate constants (R1), spin-spin relaxation rate constants (R2), and steady-state heteronuclear {1H}-15N NOEs have been determined for 69 of the 86 (excluding two prolines and the N-terminal residue) backbone amide 15N at a magnetic field strength of 14.1 Tesla. The primary relaxation data have been analyzed by using the model-free formalism of molecular dynamics, using both isotropic and axially symmetric diffusion of the molecule, to determine the overall rotational correlation time (τ(m)), the generalized order parameter (S2), the effectire correlation time for internal motions (τ(e)), and NH exchange broadening contributions (R(ex)) for each residue. As per the axially symmetric diffusion, the ratio of diffusion rates about the unique and perpendicular axes (D(is parallel with)/D(is perpendicular to)) is 0.82 ± 0.03. The two results have only marginal differences. The relaxation data have also been used to map reduced spectral densities for the NH vectors of these residues at three frequencies: 0, ω(H), and ω(N), where ω(H,N) are proton and nitrogen Larmor frequencies. The value of τ(m) obtained from model-free analysis of the relaxation data is 5.2 ns. The reduced spectral density analysis, however, yields a value of 5.7 ns. The τ(m) determined here is different from that calculated previously from time-resolved fluorescence data (4.1 ns). The order parameter ranges from 0.68 to 0.98, with an average value of 0.85 ± 0.02. A comparison of the order parameters with the X-ray B-factors for the backbone nitrogens of wild-type barstar does not show any considerable correlation. Model-free analysis of the relaxation data for seven residues required the inclusion of an exchange broadening term, the magnitude of which ranges from 2 to 9.1 s-1, indicating the presence of conformational averaging motions only for a small subset of residues. (C) 2000 Wiley-Liss, Inc.