Department of Molecular, Cellular, and Developmental Biology, University of Colorado Boulder
Probing the energetics and hidden dynamics of bacteriorhodopsin by AFM
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.
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