Probing the energetics and hidden dynamics of bacteriorhodopsin by AFM

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The forces and energetics that stabilize membrane proteins remain elusive to precise quantification. Single-molecule force spectroscopy can yield kinetic rate constants, energetics, intermediate states, unfolding pathways, and even a projection of the underlying free-energy landscape. Using recently developed micromachined AFM cantilevers, we reexamined the unfolding of individual molecules of bacteriorhodopsin (bR) embedded in its native lipid bilayer with a 100-fold improvement in time resolution and a 10-fold improvement in force precision. Numerous newly detected intermediates—many separated by as few as 2–3 amino acids—exhibited complex dynamics, including frequent refolding and state occupancies of <10 µs. Rapid and reversible dynamics in the initial unfolding of bR allowed us to measure the equilibrium energetics of a membrane protein in its native lipid bilayer, an advance over traditional results obtained by chemical denaturation in nonphysiological mixed micelles.