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Protein folding, protein collapse, and tanford's transfer model: lessons from single-molecule FRET.

ABSTRACT: The essential and nontrivial role of the denatured state of proteins in their folding reaction is being increasingly scrutinized in recent years. Single molecule FRET (smFRET) experiments show that the denatured state undergoes a continuous collapse (or coil-to-globule) transition as the concentration of a chemical denaturant is decreased, suggesting that conformational entropy of the denatured state is an important part of the free energy of folding. Such observations question the validity of the classical Tanford transfer model, which suggests that the folding free energy can be understood solely based on the difference in amino acid solvation between the folded state and a fixed unfolded state. An alternative to the transfer model is obtained here from a polymer theoretical analysis of a series of published smFRET data. The analysis shows that the free energy of denatured-state collapse has a linear dependence on denaturant concentration, an outcome of the interplay between enthalpic and entropic contributions. Surprisingly, the slope of the free energy of collapse agrees very well with the respective slope of the free energy of folding. This conformity of values obtained from two very different measurements shows that it is the collapse transition in the denatured state which mediates the effect of denaturants on folding. The energetics of folding are thus governed by the competition of solvation and conformational entropy in the denatured state.

SUBMITTER: Ziv G

PROVIDER: S-EPMC2663972 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Protein folding, protein collapse, and tanford's transfer model: lessons from single-molecule FRET.

Ziv Guy G Haran Gilad G

Journal of the American Chemical Society 20090301 8

The essential and nontrivial role of the denatured state of proteins in their folding reaction is being increasingly scrutinized in recent years. Single molecule FRET (smFRET) experiments show that the denatured state undergoes a continuous collapse (or coil-to-globule) transition as the concentration of a chemical denaturant is decreased, suggesting that conformational entropy of the denatured state is an important part of the free energy of folding. Such observations question the validity of t ...[more]

PMID: 19239269

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Project description:We describe the theory, experiment, and analysis of three-color Förster resonance energy transfer (FRET) spectroscopy for probing conformational dynamics of a fast-folding protein, α3D. In three-color FRET, site-specific labeling of fluorophores is required to avoid ambiguity resulting from various species with different combinations of labeling positions. To this end, we first attached two dyes to a cysteine residue and an unnatural amino acid and then appended a cysteine residue to the C-terminus of the protein by the sortase-mediated ligation for attaching the third dye. To determine all three FRET efficiencies, we used alternating excitation of the donor and acceptor 1 with two picosecond-pulsed lasers. Since the folded and unfolded states are not distinguishable in binned fluorescence trajectories due to fast-folding on a millisecond time scale, we used a maximum likelihood method that analyzes photon trajectories without binning the data. The extracted kinetic parameters agree very well with the previously measured parameters for the same protein with two-color FRET, suggesting that the addition of the third fluorophore does not affect the folding dynamics of the protein. From the extracted fractions of acceptor photon counts, the FRET efficiencies for all three dye pairs were calculated after various corrections. They were compared with the FRET efficiencies obtained from the global analysis of two-color segments collected in the same experiment. The FRET efficiencies of the folded state from the three-color segments agree with those from the two-color segments, whereas the three-color and two-color FRET efficiencies of the unfolded state are different. This happens because fluctuations of all three interdye distances contribute to the FRET efficiency measured in three-color FRET. We show that this difference can be accounted for by using the Gaussian chain model for the unfolded state with the parameters obtained from the analysis of two-color segments. This result shows that three-color FRET provides additional information on the flexibility of molecules that cannot be obtained from a combination of two-color FRET experiments with three dye pairs. Using the delay times of photons from the laser pulse, fluorescence lifetimes were determined using the maximum likelihood analysis. The correlation between FRET efficiencies and lifetimes of the donor, acceptor 1, and acceptor 2 was visualized in two-dimensional FRET efficiency-lifetime histograms. These histograms can be used to demonstrate the presence of conformational dynamics in a protein.

Project description:Recently developed statistical methods by Gopich and Szabo were used to extract folding and unfolding rate coefficients from single-molecule Förster resonance energy transfer (FRET) data for proteins with kinetics too fast to measure waiting time distributions. Two types of experiments and two different analyses were performed. In one experiment bursts of photons were collected from donor and acceptor fluorophores attached to a 73-residue protein, α(3)D, freely diffusing through the illuminated volume of a confocal microscope system. In the second, the protein was immobilized by linkage to a surface, and photons were collected until one of the fluorophores bleached. Folding and unfolding rate coefficients and mean FRET efficiencies for the folded and unfolded subpopulations were obtained from a photon by photon analysis of the trajectories using a maximum likelihood method. The ability of the method to describe the data in terms of a two-state model was checked by recoloring the photon trajectories with the extracted parameters and comparing the calculated FRET efficiency histograms with the measured histograms. The sum of the rate coefficients for the two-state model agreed to within 30% with the relaxation rate obtained from the decay of the donor-acceptor cross-correlation function, confirming the high accuracy of the method. Interestingly, apparently reliable rate coefficients could be extracted using the maximum likelihood method, even at low (<10%) population of the minor component where the cross-correlation function was too noisy to obtain any useful information. The rate coefficients and mean FRET efficiencies were also obtained in an approximate procedure by simply fitting the FRET efficiency histograms, calculated by binning the donor and acceptor photons, with a sum of three-Gaussian functions. The kinetics are exposed in these histograms by the growth of a FRET efficiency peak at values intermediate between the folded and unfolded peaks as the bin size increases, a phenomenon with similarities to NMR exchange broadening. When comparable populations of folded and unfolded molecules are present, this method yields rate coefficients in very good agreement with those obtained with the maximum likelihood method. As a first step toward characterizing transition paths, the Viterbi algorithm was used to locate the most probable transition points in the photon trajectories.

Dataset Information

Protein folding, protein collapse, and tanford's transfer model: lessons from single-molecule FRET.

Publications

Protein folding, protein collapse, and tanford's transfer model: lessons from single-molecule FRET.

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