Backbone dynamics of barstar: A N-15 NMR relaxation study

Backbone dynamics of uniformly N-15-labeled barstar have been studied at 32 degreesC, pH 6,7, by using N-15 relaxation data obtained from proton-detec ted 2D {H-1}-N-15 NMR spectroscopy. N-15 spin-lattice relaxation rate const ants (R-1), spin-spin relaxation rate constants (R-2), and steady-state het eronuclear {H-1}-N-15 NOEs have been determined for 69 of the 86 (excluding two prolines and the N-terminal residue) backbone amide N-15 at a magnetic held strength of 14.1 Tesla, The primary relaxation data have been analyze d by using the model-free formalism of molecular dynamics, using both isotr opic and axially symmetric diffusion of the molecule, to determine the over all rotational correlation time (tau (m)), the generalized order parameter (S-2), th, effective correlation time for internal motions (tau (e)), and N H exchange broadening contributions (R-ex) for each residue. As per the axi ally symmetric diffusion, the ratio of diffusion rates about the unique and perpendicular axes (D-parallel to/D-perpendicular to) is 0.82 +/- 0.03, Th e two results have only marginal differences. The relaxation data have also been used to map reduced spectral densities for the NH vectors of these re sidues at three frequencies: 0, omega (H), and omega (N), where w(H),(N) ar e proton and nitrogen Larmor frequencies. The value of tau (m), Obtained fr om model-free analysis of the relaxation data is 5.2 ns, The reduced spectr al density analysis, however, yields a value of 5.7 ns, The tau (m) determi ned here is different from that calculated previously from time-resolved fl uorescence data (4.1 ns), The order parameter ranges from 0.68 to 0,98, wit h an average value of 0.85 +/- 0,02, A comparison of the order parameters w ith the X-ray B-factors for the backbone nitrogens of wild-type barstar doe s not show any considerable correlation. Model-free analysis of the relaxat ion data for seven residues required the inclusion of an exchange broadenin g term, the magnitude of which ranges from 2 to 9.1 s(-1), indicating the p resence of conformational averaging motions only for a small subset of resi dues. Proteins 2000;41:460-474, (C) 2000 Wiley-Liss, Inc.