Quantifying the Native Energetics Stabilizing Bacteriorhodopsin by Single-Molecule Force Spectroscopy

We quantified the equilibrium (un)folding free energy $\mathrm{\Delta }{G}_0$ of an eight-amino-acid region starting from the fully folded state of the model membrane-protein bacteriorhodopsin using single-molecule force spectroscopy. Analysis of equilibrium and nonequilibrium data yielded consistent, high-precision determinations of $\mathrm{\Delta }{G}_0$ via multiple techniques (force-dependent kinetics, Crooks fluctuation theorem, and inverse Boltzmann analysis). We also deduced the full 1D projection of the free-energy landscape in this region. Importantly, $\mathrm{\Delta }{G}_0$ was determined in bacteriorhodopsin’s native bilayer, an advance over traditional results obtained by chemical denaturation in nonphysiological detergent micelles.