Localized thermodynamic coupling between hydrogen bonding and microenvironment polarity substantially stabilizes proteins

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Abstract
The contribution of hydrogen bonding to the thermodynamics of protein folding is not well understood. The strength of hydrogen bonds is now found to depend on the polarity of their microenvironment, being stronger in non-polar surroundings. Thus, the burial or solvent exposure of a few hydrogen bonds near the surface of a protein can significantly stabilize or destabilize its native state. The energetic contributions of hydrogen bonding to protein folding are still unclear, despite more than 70 years of study. This is due partly to the difficulty of extracting thermodynamic information about specific interactions from protein mutagenesis data and partly to the context dependence of hydrogen bond strengths. Herein, we test the hypothesis that hydrogen bond strengths depend on the polarity of their microenvironment, with stronger hydrogen bonds forming in nonpolar surroundings. Double-mutant cycle analysis using a combination of amide-to-ester backbone mutagenesis and traditional side chain mutagenesis revealed that hydrogen bonds can be stronger by up to 1.2 kcal mol−1 when they are sequestered in hydrophobic surroundings than when they are solvent exposed. Such large coupling energies between hydrogen bond strengths and local polarity suggest that the context dependence of hydrogen bond strengths must be accounted for in any comprehensive account of the forces responsible for protein folding.