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 (ΔΔG). It has been applied to >80 different residues of bacteriorhodopsin (bR), a model membrane protein. However, such experiments have several key limitations: (i) a non-native lipid environment, (ii) a denatured state with significant secondary structure, (iii) error introduced by extrapolation to zero denaturant, and (iv) 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-ms 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 ΔΔG, 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 ΔΔG for mutant L223A (−2.3 ± 0.6 kcal/mol) quantitatively agreed with past chemical-denaturation results while our ΔΔG for mutant V217A was 2.2-fold larger (−2.4 ± 0.6 kcal/mol). We attribute the latter result, in part, to contact between Val²¹⁷ 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 ΔΔG for a fully folded membrane protein embedded in its native bilayer.

All data are comma delimited and in SI units. Data shown in each figure in the paper or SI Appendix are saved as separate files in this repository. Force-spectroscopy data are saved in the form of force versus either time or molecular extension ("ext"). Rate map data are denoted by molecular transition; e.g,. k_01 is the rate for the transition from state 0 to state 1. Uncertainties in these rates are saved as, e.g., dk_01. These rates are to be plotted versus force_data. Rates evaluated from Bell-model fits are denoted, e.g., fitBE_01, to be plotted versus force_fitBE. Image data are stored as two-dimensional arrays of numerical values, each of which is a height.