Backbone Hydrogen Bond Strengths Can Vary Widely in Transmembrane Helices

[Image: see text] Although backbone hydrogen bonds in transmembrane (TM) helices have the potential to be very strong due to the low dielectric and low water environment of the membrane, their strength has never been assessed experimentally. Moreover, variations in hydrogen bond strength might be necessary to facilitate the TM helix breaking and bending that is often needed to satisfy functional imperatives. Here we employed equilibrium hydrogen/deuterium fractionation factors to measure backbone hydrogen bond strengths in the TM helix of the amyloid precursor protein (APP). We find an enormous range of hydrogen bond free energies, with some weaker than water–water hydrogen bonds and some over 6 kcal/mol stronger than water–water hydrogen bonds. We find that weak hydrogen bonds are at or near preferred γ-secretase cleavage sites, suggesting that the sequence of APP and possibly other cleaved TM helices may be designed, in part, to make their backbones accessible for cleavage. The finding that hydrogen bond strengths in a TM helix can vary widely has implications for membrane protein function, dynamics, evolution, and design.