Quantifying the Initial Unfolding of Bacteriorhodopsin Reveals Retinal Stabilization

The forces that stabilize membrane proteins remain elusive to precise quantification. Particularly important but poorly resolved are the forces present during a membrane protein's initial unfolding, where the most native set of interactions are present. We developed a high‐precision, atomic force microscopy assay to study the initial unfolding of bacteriorhodopsin. We discovered rapid near‐equilibrium folding between the first three unfolding states that corresponded to the unfolding of 5 and 8 amino‐acids respectively when using a cantilever optimized for 2‐µs resolution. Interestingly, 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 >1 min. We expect that this ability to measure the rapid and reversible dynamics in the initial unfolding of bacteriorhodopsin provides a platform for quantifying the energetics of membrane proteins under native‐like conditions.

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