It has been traditionally difficult to measure the thermodynamic stability of membrane proteins because fully reversible protocols for complete folding these proteins were not available. Knowledge of the thermodynamic stability of membrane proteins is desirable not only from a fundamental theoretical standpoint, but is also of enormous practical interest for the rational design of membrane proteins and for optimizing conditions for their structure determination by crystallography or NMR. Here, we describe the design of a fully reversible system to study equilibrium folding of the outer membrane protein A from Escherichia coli in lipid bilayers. Folding is shown to be two-state under appropriate conditions permitting data analysis with a classical folding model developed for soluble proteins. The resulting free energy and m value, i.e., a measure of cooperativity, of unfolding are and m = 1.1 kcal/mol M–1, respectively, in a reference bilayer composed of palmitoyl-oleoyl-phosphatidylcholine (C16:0C18:1PC) and palmitoyloleoyl-phosphatidylglycerol (C16:0C18:1PG). These values are strong functions of the lipid bilayer environment. By systematic variation of lipid headgroup and chain composition, we show that elastic bilayer forces such as curvature stress and hydrophobic mismatch modulate the free energy and cooperativity of folding of this and perhaps many other membrane proteins.
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