Protein folding is a central life process. Errors in folding are the cause of many diseases and an understanding of protein folding is needed for wide ranging fields such as protein design, evolution, and drug design. Consequently, massive efforts have been directed at various aspects of folding. In the quest to understand protein folding, however, membrane proteins have been largely ignored. Membrane protein folding must be brought up to speed. We propose to learn more about the unfolded state in bilayers, the process of membrane protein folding, and how the final structure is defined. We will emerge from the proposed studies with new technologies for membrane protein folding studies, and with a much improved picture of how membrane proteins fold in true bilayer environments.
The specific aims are:
Aim I. (Folded state) Investigate the role of H-bonds in transmembrane helix bending. In the membrane where H-bonds can be very strong, mechanisms exist to allow transmembrane helices to bend. Helix bending is a fundamental feature of membrane protein structure and dynamics that must be understood. To investigate bending mechanisms we will employ a new method we have developed to measure backbone H-bond energies and observe how they correlate with sequence and structural changes.
Aim II. (Folded state) How do changes in bilayer properties affect membrane protein stability? Lipid composition varies in different membrane domains, during signaling and as the result of disease. These changes can have profound effects on membrane protein function. Yet there are still no quantitative studies of bilayer composition effects on the stability of helical membrane proteins. We now have a method to attack this problem and will seek to fill in the gap in our knowledge.
Aim III. (Folding) How do membrane proteins find the native state? The nature of the search toward the folded state remains largely uncharacterized for helical membrane proteins. We will characterize the folding transition state of a helical membrane protein in micelles for the first time, and develop methods for characterizing the folding process in bilayers.
Aim I V. (Unfolded State) The structure of the unfolded state in membranes. The nature of the unfolded state of membrane proteins in bilayers is largely unknown. We now have a method for trapping the unfolded state in bilayers. We will characterize the structure of this unfolded state using EPR methods, thereby helping to complete a basic picture of membrane protein folding.

Public Health Relevance

The genome is manifest in part by the protein molecules it encodes. These proteins are often designed to fold up into a unique structure that is essential for its biological role. Disease can occur if the folding process is disrupted by mutation or other physiological processes. We are working to understand how the large class of proteins that float in cell membranes manage to assemble so that we can learn how to intervene in folding diseases.

National Institute of Health (NIH)
Research Project (R01)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Chin, Jean
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University of California Los Angeles
Schools of Arts and Sciences
Los Angeles
United States
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Cao, Zheng; Bowie, James U (2014) An energetic scale for equilibrium H/D fractionation factors illuminates hydrogen bond free energies in proteins. Protein Sci 23:566-75
Hong, Heedeok; Chang, Yu-Chu; Bowie, James U (2013) Measuring transmembrane helix interaction strengths in lipid bilayers using steric trapping. Methods Mol Biol 1063:37-56
Bowie, James U (2013) Structural biology. Membrane protein twists and turns. Science 339:398-9
Nam, Hyun-Jun; Han, Seong Kyu; Bowie, James U et al. (2013) Rampant exchange of the structure and function of extramembrane domains between membrane and water soluble proteins. PLoS Comput Biol 9:e1002997
Jefferson, Robert E; Blois, Tracy M; Bowie, James U (2013) Membrane proteins can have high kinetic stability. J Am Chem Soc 135:15183-90
Schlebach, Jonathan P; Cao, Zheng; Bowie, James U et al. (2012) Revisiting the folding kinetics of bacteriorhodopsin. Protein Sci 21:97-106
Hong, Heedeok; Bowie, James U (2011) Dramatic destabilization of transmembrane helix interactions by features of natural membrane environments. J Am Chem Soc 133:11389-98
Schlebach, Jonathan P; Kim, Moon-Soo; Joh, Nathan H et al. (2011) Probing membrane protein unfolding with pulse proteolysis. J Mol Biol 406:545-51
Bowie, James U (2011) Membrane protein folding: how important are hydrogen bonds? Curr Opin Struct Biol 21:42-9
Meruelo, Alejandro D; Samish, Ilan; Bowie, James U (2011) TMKink: a method to predict transmembrane helix kinks. Protein Sci 20:1256-64

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