摘要： 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.