Improving AFM Reveals a Multitude of Hidden Dynamics in the Unfolding of a Membraneprotein

Abstract

Protein folding occurs as a set of transitions between structural states within an energy landscape. An oversimplified view of the folding process emerges when transiently populated states are undetected because of limited instrumental resolution. Moreover, predicting the structure and stability of membrane proteins significantly lags success with globular protein. Using force spectroscopy optimized for 1-µs resolution, we reexamined the unfolding of individual bacteriorhodopsin (BR) molecules in native lipid bilayers with a 100-fold improvement in time resolution and a 10-fold improvement in force precision. The resulting data revealed the unfolding pathway in unprecedented detail. 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. To elucidate the detailed dynamics of individual membrane proteins under their most native-likeset of intra- and inter-helix contacts, we labeled BR at its c-terminal with a copper-free click chemistry reagent and then gently but covalently coupled BR to an azide-functionalized, PEG-coated tip. This process suppressed nonspecific adhesion that confounds analysis of the initial unfolding of BR. The resulting force-extension curves revealed rapid near-equilibrium folding dynamics between previously undetected intermediate states. We assigned the first state to the top of the G-helix with the second and third states corresponding to a total unfolding of 5 and 9 amino acids. Interestingly, this assignment placed the third state where BR's retinal binds to lysine 216. We verified this assignment by performing dynamic force spectroscopy on BR with and without its retinal. We also reconstructed the full 1D free-energy landscape underlying the initial 9 amino acid unfolding of BR. Looking forward, this reversible unfolding and refolding assay should provide a platform to precisely quantify the energetics of membrane folding under native-like conditions.