Hydrogen Bonding and Equilibrium Protium−Deuterium Fractionation Factors in the Immunoglobulin G Binding Domain of Protein G

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Abstract
Protium−deuterium fractionation factors (φ) were determined for more than 85% of the backbone amide protons in the IgG binding domains of protein G, GB1 and GB2, from NMR spectra recorded over a range of H2O/D2O solvent ratios. Previous studies suggest a correlation between φ and hydrogen bond strength; amide and hydroxyl groups in strong hydrogen bonds accumulate protium (φ < 1), while weak hydrogen bonds accumulate deuterium (φ > 1). Our results show that the α-helical residues have slightly lower φ values (1.03 ± 0.05) than β-sheet residues (1.12 ± 0.07), on average. The lowest φ value obtained (0.65) does not involve a backbone amide but rather is for the interaction between two side chains, Y45 and D47. Fractionation factors for solvent-exposed residues are between the α-helix and β-sheet values, on average, and are close to those for random coil peptides. Further, the difference in φav between α-helix and solvent-exposed residues is small, suggesting that differences in hydrogen bond strength for intrachain hydrogen bonds and amide···water hydrogen bonds are also small. Overall, the enrichment for deuterium suggests that most backbone···backbone hydrogen bonds are weak.