Modest stabilization by most hydrogen-bonded side-chain interactions in membrane proteins

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Abstract
Hydrogen bonds have been widely assumed to be strongly stabilizing in membrane proteins, but there have been few experimental tests of this notion. In this work, interaction free energies were measured between eight hydrogen-bonded side chains in bacteriorhodopsin. Contrary to expectations, most make only modest stabilizing contributions, averaging 0.6 kcal mol−1. The results suggest that views of membrane protein folding, evolution and function should reflect such weak polar side-chain interactions. Hydrogen bonds have been widely assumed to be strongly stabilizing in membrane proteins. But in this work, interaction free energies were measured between eight hydrogen bonded side chains in bacteriorhodopsin and it was found that most make only modest stabilizing contributions. This suggests that views of membrane protein folding, evolution and function should reflect such weak polar side chain interactions. Understanding the energetics of molecular interactions is fundamental to all of the central quests of structural biology including structure prediction and design, mapping evolutionary pathways, learning how mutations cause disease, drug design, and relating structure to function. Hydrogen-bonding is widely regarded as an important force in a membrane environment because of the low dielectric constant of membranes and a lack of competition from water1,2,3,4,5,6. Indeed, polar residue substitutions are the most common disease-causing mutations in membrane proteins6,7. Because of limited structural information and technical challenges, however, there have been few quantitative tests of hydrogen-bond strength in the context of large membrane proteins. Here we show, by using a double-mutant cycle analysis, that the average contribution of eight interhelical side-chain hydrogen-bonding interactions throughout bacteriorhodopsin is only 0.6 kcal mol-1. In agreement with these experiments, we find that 4% of polar atoms in the non-polar core regions of membrane proteins have no hydrogen-bond partner and the lengths of buried hydrogen bonds in soluble proteins and membrane protein transmembrane regions are statistically identical. Our results indicate that most hydrogen-bond interactions in membrane proteins are only modestly stabilizing. Weak hydrogen-bonding should be reflected in considerations of membrane protein folding, dynamics, design, evolution and function.