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.
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We thank the staff at the beamlines 8.2.1 and 8.2.2 at the Advanced Light Source; F. Pettit for advice on statistics; M. Philips for assisting with circular dichroism experiments; S. Bassilian for assisting with LC–MS analysis of intact bacteriorhodopsin; Y. Ihm for the identification of transmembrane regions; Z. Zhang for providing deuterium-exchange data reduction software MAGTRAN and LAPLACE; N. L. Kelleher for providing the acid-labile detergent; and M. Chamberlain, H. Cheng, E. Gendel, H. Hong, Y. Ihm, A. D. Meruelo, T. Mitchell and R. Stafford for critically reading the manuscript. This work was supported by National Institutes of Health grant RO1 GM063919 (J.U.B.) and by the National Institutes of Health National Cancer Institute Innovative Molecular Analysis Technologies Program (V.L.W.).
Author Contributions N.H.J. and J.U.B. designed the research and prepared the manuscript. N.H.J. performed the vast majority of the experiments and structure analyses. A.M. and D.Y. assisted with mutagenesis and protein purification. A.M. crystallized the T90A/D115A mutant. S.F. collected and processed some diffraction data and helped with structure determination and refinement. J.P.W. assisted site-directed mutagenesis verification by LC–MS analysis of intact bacteriorhodopsin, provided technical advice on mass spectrometry, and helped develop the H/D exchange method. V.L.W. assisted in H/D exchange data analysis, including the provision of specialized software and hardware, and provided help with H/D exchange methods.
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Joh, N., Min, A., Faham, S. et al. Modest stabilization by most hydrogen-bonded side-chain interactions in membrane proteins. Nature 453, 1266–1270 (2008). https://doi.org/10.1038/nature06977
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