NMR relaxation mechanisms for backbone carbonyl carbons in a C-13, N-15-labeled protein

The predominant relaxation mechanisms for backbone carbonyl carbon (C-13') relaxation in a C-13, N-15-doubly enriched sample of the thermostable Sso7d protein have been investigated. Pulse sequences for measurements of longitudinal and transverse C-13' relaxation rates were implemented, and these rates were measured at magnetic fields of 11.7 and 14.1 T. The field dependence in measured rates is small and consistent with a predominant contribution from chemical-shift anisotropy (CSA) to C-13' relaxation. A pulse sequence for measurement of {H-1} - C-13' cross-relaxation rates (steady-state NOEs) was also developed. This experiment reveals a significant NOE between protons and all C-13', indicating that dipolar interactions between these nuclei contribute to C-13' relaxation. Experiments designed to suppress cross correlation between CSA relaxation and dipole-dipole (DD) relaxation due to neighboring C-13(alpha) indicate that this effect is negligible. A more quantitative treatment is also presented, in which backbone dynamics parameters are fitted to average C-13' relaxation rates using Lipari-Szabo expressions for the spectral density. This fit, which reproduces well expected backbone dynamics parameters for a folded protein, is used to estimate the relative contributions of various mechanisms to C-13' relaxation. It is found that both longitudinal and transverse relaxation rates are dominated by CSA relaxation and contain significant contributions due to DD relaxation induced by nearby protons. Contributions from DD relaxation due to covalently bound C-13(alpha) and N-15 are comparably small. The predominant effects of CSA and H-1-C-13' DD interactions, for which physical and geometrical parameters are uncertain, complicate the use of C-13' relaxation as a sequence-specific probe for protein backbone dynamics. (C) 1997 Academic Press.