Protein folding is inherently a heterogeneous process because of the very large number of microscopic pathways that connect the myriad unfolded conformations to the unique conformation of the native structure. In a first step towards the long-range goal of describing the distribution of pathways experimentally, Förster resonance energy transfer1 (FRET) has been measured on single, freely diffusing molecules2,3,4. Here we use this method to determine properties of the free-energy surface for folding that have not been obtained from ensemble experiments. We show that single-molecule FRET measurements of a small cold-shock protein expose equilibrium collapse of the unfolded polypeptide and allow us to calculate limits on the polypeptide reconfiguration time. From these results, limits on the height of the free-energy barrier to folding are obtained that are consistent with a simple statistical mechanical model, but not with the barriers derived from simulations using molecular dynamics. Unlike the activation energy, the free-energy barrier includes the activation entropy and thus has been elusive to experimental determination for any kinetic process in solution.
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We thank A. Szabo and I. Gopich for suggestions and guidance on theoretical issues; S. Doniach, J. Hofrichter and G. Hummer for discussion and comments on the manuscript; L. Davis and R. Zare for advice regarding the single-molecule instrument; and L. Pannell for mass spectroscopy measurements. B.S. was supported by an Emmy Noether fellowship from the Deutsche Forschungsgemeinschaft.
The authors declare that they have no competing financial interests.
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Schuler, B., Lipman, E. & Eaton, W. Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy. Nature 419, 743–747 (2002). https://doi.org/10.1038/nature01060
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