Hydrogen bond geometry and H-2/H-1 fractionation in proteins

Measurement of H-2/H-1 equilibrium exchange (Phi) is commonly applied as an indirect probe of hydrogen bonds in small molecules. To expand the limited set of protein Phi measurements and to gain insight into the putative corr elation between low Phi, and hydrogen bond strength, we report measurements for two proteins, src SH3 and ubiquitin. A hydrogen bond network in Gallus gallus src SH3 domain, involving residues 30, 47, and 50, contributes simi lar to 2.0 kcal/mol to native state stability and provides an excellent sys tem in which to test the purported connection between hydrogen bonding netw orks and protium enrichment. All observed sites in src SH3 had fractionatio n factors greater than unity. In particular, the backbone protons associate d with the hydrogen bond network at residues Glu30, Ser47;, and Thr50 all e xhibit moderate deuterium enrichment. Measured fractionation values in ubiq uitin range from Phi = 1.52 for the amide of residue Ser20 to a remarkably low Phi = 0.29 for the amide of Thr9. The majority (75%) of backbone amides fall between 0.9 < Phi < 1.3, with an average Phi = 1.07, closely matching the average Phi = 1.11 previously determined by another triple resonance m ethod, and consistent with the fractionation observed in other weak hydroge n bonding amide systems. A survey of protein sites exhibiting low fractiona tion show a conservation of hydrogen bonding geometry. Our data, in combina tion with other studies, suggest that H-1/H-2 fractionation at protein back bone amides is a product of the complex three dimensional and static protei n hydrogen bonding environment that restricts or enhances specific vibratio nal modes and is largely independent of hydrogen bonding strength.