Solution NMR Characterization of Hydrogen Bonds in a Protein by Indirect Measurement of Deuterium Quadrupole Couplings

摘要

Hydrogen bonds stabilize protein and nucleic acid structure, but little direct spectroscopic data have been available for characterizing these critical interactions in biological macromolecules. It is demonstrated that the electric field gradient at the nucleus of an amide hydrogen can be determined residue-specific by measurement of15N NMR relaxation times in proteins dissolved in D2O, and uniformly enriched with13C and15N. In D2O, all backbone amide protons can be exchanged with solvent deuterons, and theT1relaxation rate of a deuteron is dominated by its quadrupole coupling constant (QCC), which is directly proportional to the electric field gradient at the nucleus.2HNT1relaxation can be measured quantitatively through its effect on theT2relaxation of its directly attached15N. QCC values calculated from2HNT1and previously reported spectral densities correlate with the inverse cube of the X-ray crystal structure-derived hydrogen bond lengths: QCC = 228 + Σi130 cos αi/ri3kHz, where α is the N–H···Oiangle andris the backbone–backbone (N–)H···Oi(=C) hydrogen bond distance in ?ngstroms.