The molecular yo-yo method: Live jump detection improves throughput of single-molecule force spectroscopy for out-of-equilibrium transitions

摘要

By monitoring multiple molecular transitions, force-clamp andtrap-position-clamp methods have led to precise determinations of the freeenergies and free energy landscapes for molecular states populated inequilibrium at the same or similar forces. Here, we present a powerful newelaboration of the force-clamp and force-jump methods, applicable totransitions far from equilibrium. Specifically, we have implemented a live jumpdetection and force-clamp algorithm that intelligently adjusts and maintainsthe force on a single molecule in response to the measured state of thatmolecule. We are able to collect hundreds of individual molecular transitionsat different forces, many times faster than previously, permitting us toaccurately determine force-dependent lifetime distributions and reaction rates.Application of our method to unwinding and rewinding the nucleosome inner turn,using optical tweezers reveals experimental lifetime distributions thatcomprise a statistically-meaningful number of transitions, and that areaccurately single exponential. These measurements significantly reduce theerror in the previously measured rates, and demonstrate the existence of asingle, dominant free energy barrier at each force studied. A key benefit ofthe molecular yoyo method for nucleosomes is that it reduces as far as possiblethe time spent in the tangentially-bound state, which minimizes the loss ofnucleosomes by dissociation.