Free-energy changes of bacteriorhodopsin point mutants measured by single-molecule force spectroscopy.

Single amino acid mutations provide quantitative insight into the energetics that underlie the dynamics and folding of membrane proteins. Chemical denaturation is the most widely used assay and yields the change in unfolding free energy (ΔΔ). It has been applied to >80 different residues of bacteriorhodopsin (bR), a model membrane protein. However, such experiments have several key limitations: 1) a nonnative lipid environment, 2) a denatured state with significant secondary structure, 3) error introduced by extrapolation to zero denaturant, and 4) the requirement of globally reversible refolding. We overcame these limitations by reversibly unfolding local regions of an individual protein with mechanical force using an atomic-force-microscope assay optimized for 2 μs time resolution and 1 pN force stability. In this assay, bR was unfolded from its native bilayer into a well-defined, stretched state. To measure ΔΔ, we introduced two alanine point mutations into an 8-amino-acid region at the C-terminal end of bR's G helix. For each, we reversibly unfolded and refolded this region hundreds of times while the rest of the protein remained folded. Our single-molecule-derived ΔΔ for mutant L223A (-2.3 ± 0.6 kcal/mol) quantitatively agreed with past chemical denaturation results while our ΔΔ for mutant V217A was 2.2-fold larger (-2.4 ± 0.6 kcal/mol). We attribute the latter result, in part, to contact between Val and a natively bound squalene lipid, highlighting the contribution of membrane protein-lipid contacts not present in chemical denaturation assays. More generally, we established a platform for determining ΔΔ for a fully folded membrane protein embedded in its native bilayer.