Abstract
The presence and extent of hydrogen-bonding (H-bonding) cooperativity in proteins remains a fundamental question, which in the past has been studied extensively, mostly by infrared and fluorescence measurements on model peptides. We demonstrate that such cooperativity can be studied in an intact protein by hydrogen/deuterium exchange NMR spectroscopy. The method is based on the fact that substitution of NH by ND in a backbone amide group slightly weakens the N-H···O═C hydrogen bond. Our results show that such substitution at position i in an α-helix impacts the (1)H and (15)N chemical shifts of the amide sites of residues i - 3 to i + 3. Quantum mechanical calculations indicate that the upfield shifts of (1)H and (15)N resonances at site i, observed upon H/D exchanges at sites i - 3, i + 1, i + 2, and i + 3, correspond to a decrease of the ith backbone amide electric dipole moment, which weakens its H-bonding and long-range electrostatic interactions with other backbone amides in the α-helix. These results provide new quantitative insights into the cooperativity of H-bonding in protein α-helices.