Hydrogen bonds stabilize protein and nucleic acid structure, but recent results indicate that the amide proton chemical shift little direct spectroscopic data have been available for characterizanisotropy (CSA) varies strongly with hydrogen bond ing these critical interactions in biological macromolecules. It is strength (8). Chemical shifts are also influenced by other demonstrated that the electric field gradient at the nucleus of an geometric and ring current effects, and it remains difficult amide hydrogen can be determined residue-specific by measureto separate out the effect of hydrogen bonding, at least for ment of 15 N NMR relaxation times in proteins dissolved in D 2 O, the isotropic shift (9). and uniformly enriched with 13 C and 15 N. In D 2 O, all backbone
As the hydrogen bond is electrostatic in nature, an ideal amide protons can be exchanged with solvent deuterons, and the probe of this interaction reports on its local electric environ-T 1 relaxation rate of a deuteron is dominated by its quadrupole ment. Deuterium possesses an electric quadrupole moment coupling constant (QCC), which is directly proportional to the and therefore is exquisitely sensitive to its local electric envielectric field gradient at the nucleus. 2 H N T 1 relaxation can be measured quantitatively through its effect on the T 2 relaxation of ronment. There are no complications from Sternheimer its directly attached 15 N. QCC values calculated from 2 H N T 1 and shielding effects (10, 11), and since its single valence is previously reported spectral densities correlate with the inverse spherical, field gradients result primarily from the electrons cube of the X-ray crystal structure-derived hydrogen bond lengths: and nuclei of nearby atoms. Qualitatively, a short N-QCC Å 228 / ͚ i 130 cos a i /r 3 i kHz, where a is the N-HrrrO i 2 HrrrO|C hydrogen bond results in a more symmetrical angle and r is the backbone-backbone (N-)HrrrO i (|C) hyelectrical environment at the deuterium nucleus relative to drogen bond distance in a ˚ngstroms. ᭧ 1997 Academic Press a long hydrogen bond. That is, shorter hydrogen bonds will result in weaker electric field gradients and, therefore, smaller 2 H quadrupolar coupling constant (QCC) values.