Backbone dynamics of barstar: a 15N 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 effective 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_||/D ) 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.