Abstract
The transition-path is the tiny fraction of an equilibrium, single-molecule trajectory when the free-energy barrier is actually crossed. For a two-state protein-folder, the transition-path contains all the mechanistic information on how a protein folds. However, because it is so fast a transition-path has never been observed experimentally for any molecular system in the condensed phase. As a first step toward observing transition-paths, we are using single molecule FRET to measure average transition-path times (tTP). Using the Gopich/Szabo maximum-likelihood method to analyze photon trajectories, we have now determined tTP's for all-beta (FBP28-WW domain, tTP=2μs) and all-alpha proteins (α3D, tTP=12 μs), and an upper bound for an alpha/beta protein (protein G, tTP <10 μs) (Chung et al., Science 2012; Chung, Eaton, Nature, 2013). The surprising result is that the folding times for the WW domain and protein G differ by 10,000-fold, yet the tTP's differ by less than 5-fold, so a fast and slow-folding protein take almost the same time when they actually fold. Szabo's theory for barrier crossing of a Brownian particle explains this result by showing that tTP is insensitive to the barrier height, i.e. tTP μ ln(3βΔG∗)/D∗. Studies of the viscosity- and temperature-dependence of tTP for α3D suggest that the extremely low viscosity-dependence (∼η0.3) arises from a lower D∗ due to increased internal friction (“rougher” energy landscape). Lowering the pH to neutralize 12 carboxylates eliminates potential salt bridges and reduces the viscosity dependence to that previously observed (∼η0.6) for other all-alpha proteins. These results provide the first glimpse of the structural origin for internal friction in protein folding, suggesting that the lower D∗ for α3D arises from making and breaking non-native salt-bridges during the transition path, as observed in MD simulations by the Shaw group (Best, Hummer, Eaton, PNAS 2013).
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have