Free-energy changes of bacteriorhodopsin point mutants measured by single-molecule force spectroscopy
David R. Jacobson, Thomas T. Perkins
Abstract
Significance Membrane proteins are of great biological and biomedical interest; they constitute 30% of encoded proteins and are the targets of ∼50% of drugs. Measurements of membrane protein energetics by mutating individual amino acids provide information that cannot be obtained from structural studies alone. Traditional thermodynamic assays employ chemical denaturants and nonnative micelles that imperfectly reflect the biologically relevant conditions of the native lipid bilayer. Additionally, chemical denaturation is not thermodynamically reversible for many proteins, limiting its scope. Here, we present a different approach that is based on the mechanical unfolding of individual membrane proteins. Our method yields precise energetic characterization of membrane protein point mutants embedded in their native lipid bilayer without introducing confounding denaturant interactions or requiring global reversibility.