Protein folding transition path times from single molecule FRET

Abstract

The transition path is the tiny segment of a single molecule trajectory when the free energy barrier between states is crossed and for protein folding contains all of the information about the self-assembly mechanism. As a first step toward obtaining structural information during the transition path from experiments, single molecule FRET spectroscopy has been used to determine average transition path times from a photon-by-photon analysis of fluorescence trajectories. These results, obtained for several different proteins, have already provided new and demanding tests that support both the accuracy of all-atom molecular dynamics simulations and the basic postulates of energy landscape theory of protein folding.

Ultrafast folding kinetics of WW domains reveal how the amino acid sequence determines the speed limit to protein folding

“…mutants of other WW domains (33). They are also consistent with the mean folding TP times derived from single-molecule fluorescence experiments on other proteins (27). These observations agree with the expectation for the molecular phase.…”

“…Such folding TPs have been recently detected using single-molecule fluorescence (24), optical tweezers (25), and atomic force microscopy (26). The somewhat limited experimental information about folding TPs obtained thus far points to a universal rate prefactor (27), with proteins designed de novo and thus not optimized by natural selection as possible exceptions (28). Here, we aim to investigate to what extent the folding rate prefactor, or speed limit, varies among naturally evolved amino acid sequences that fold onto the same native 3D structure.…”

Mapping Multiple Distances in a Multidomain Protein for the Identification of Folding Intermediates

“…A more detailed understanding of this process was obtained thanks to the development of the theory of funnel-shaped multidimensional folding energy landscapes (1). With the support of these theoretical predictions, experimental investigations based on single-molecule Förster resonance energy transfer (smFRET) spectroscopy and single molecule force spectroscopy contributed significantly to our current knowledge (2)(3)(4)(5)(6)(7). The use of smFRET offers a unique tool to map conformational changes within the protein structure, thus allowing one to observe the distribution in populations.…”

Measuring the average shape of transition paths during the folding of a single biological molecule

“…They are technically challenging to measure in folding reactions, however, because they can only be observed in single molecules and have a very brief duration. As a result, it has only recently become possible to measure transition path properties directly (3). Work to date using fluorescence and force spectroscopy has probed properties such as the average transition path time for proteins and nucleic acids (4)(5)(6)(7)(8), the variations in transit times for individual transitions (9,10), the occupancy statistics within transition paths (11,12), the distribution of velocities along the transition paths (13), and the agreement between experiment and theory for the properties measured to date (13)(14)(15).…”