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Abstract The forces that stabilize membrane proteins remain elusive to precise quantification. Particularly important, but poorly resolved, are the forces present during the initial unfolding of a membrane protein, where the most native set of interactions is present. A high‐precision, atomic force microscopy assay was developed to study the initial unfolding of bacteriorhodopsin. A rapid near‐equilibrium folding between the first three unfolding states was discovered, the two transitions corresponded to the unfolding of five and three amino acids, respectively, when using a cantilever optimized for 2 μs resolution. The third of these states was retinal‐stabilized and previously undetected, despite being the most mechanically stable state in the whole unfolding pathway, supporting 150 pN for more than 1 min. This ability to measure the dynamics of the initial unfolding of bacteriorhodopsin provides a platform for quantifying the energetics of membrane proteins under native‐like conditions.