LAUSR

Pulling apart protein unfolding Elucidating the details of how complex proteins fold is a longstanding challenge. Key insights into the unfolding pathways of diverse proteins have come from single-molecule force spectroscopy (SMFS) experiments in which proteins are literally pulled apart. Yu et al. developed a SMFS technique that could unfold individual bacteriorhodopsin molecules in a native lipid bilayer with 1-µs temporal resolution (see the Perspective by Müller and Gaub). The technique delivered a 100-fold improvement over earlier studies of bacteriorhodopsin and revealed many intermediates not seen before. The authors also observed unfolding and refolding transitions between intermediate states. Science, this issue p. 945; see also p. 907 Mechanical unfolding of a membrane protein reveals previously undetected intermediates and equilibrium refolding. 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. Using force spectroscopy optimized for 1-microsecond resolution, we reexamined the unfolding of individual bacteriorhodopsin molecules in native lipid bilayers. The experimental data reveal the unfolding pathway in unprecedented detail. Numerous newly detected intermediates—many separated by as few as two or three amino acids—exhibited complex dynamics, including frequent refolding and state occupancies of <10 μs. Equilibrium measurements between such states enabled the folding free-energy landscape to be deduced. These results sharpen the picture of the mechanical unfolding of membrane proteins and, more broadly, enable experimental access to previously obscured protein dynamics.