Experiments so far have been mainly concerned with measurements on the unfolded state of proteins since the role of the structure and dynamics of the unfolded states of proteins in determining the kinetics and mechanisms of protein folding is a relatively unexplored area. At high concentrations of chemical denaturants, polypeptides behave like random coil homopolymers and the average radius of gyration (Rg) appears to agree well with dimensional scaling laws from homopolymer theory. However, at low denaturant concentrations where proteins fold, evidence for specific structure in the unfolded state has been observed for several proteins. Several experimental studies, moreover, suggest that native-like structure in the unfolded state may play an important role in the folding mechanism.? ? Single molecule FRET is well-suited to investigate the structure and dynamics of unfolded proteins, since unlike ensemble studies it can resolve the folded and unfolded subpopulations in low denaturant regimes and also reveal information on structural distributions. Although single molecule FRET has been mostly used in qualitative structural studies, quantitative distance information has been obtained for DNA and polyproline. In the case of unfolded proteins, quantitative FRET analysis is complicated by the wide range of end-to-end distances, chain dynamics, and presence of large fluorescent dye labels. Here we demonstrate the quantitative applicability of single molecule FRET measurements to unfolded proteins using intensity and lifetime measurements in combination with molecular simulations for two well-characterized two-state proteins, the 64 residue / protein L and the 66 residue all- cold shock protein CspTm. The size distribution of the unfolded state is determined for both proteins, and for protein L is compared with equilibrium and time-resolved small angle X-ray scattering experiments. The donor fluorescence decays and FRET efficiency distribution widths are analyzed to extract dynamical information on the unfolded protein chain on the nanosecond and millisecond time scales, respectively. Finally, simulations using both simplified and all-atom protein models are used throughout this study to justify the analysis and to support and interpret the results.? ? The average radius of gyration (Rg) calculated from FRET data on freely diffusing molecules is identical for the two unfolded proteins at guanidinium chloride concentrations above 3M, and the FRET-derived Rg of protein L agrees well with the Rg previously measured by equilibrium small angle X-ray scattering. As the denaturant concentration is lowered, the mean FRET efficiency of the unfolded subpopulation increases, signaling collapse of the polypeptide chain, with protein L being slightly more compact than CspTm. A decrease in Rg with decreasing denaturant is also observed in all-atom molecular dynamics calculations in explicit water/urea solvent, and Langevin simulations of a simplified representation of the polypeptide suggest that collapse can result either from increased inter-residue attraction or decreased excluded volume. Analysis of the donor fluorescence decay of the unfolded subpopulation of both proteins gives information about the end-to-end chain distribution and suggests that chain dynamics is slow compared to the donor lifetime of 2 ns, while the bin-size independence of the small excess width above the shot noise for the FRET efficiency distributions may result from incomplete conformational averaging on even the 1 ms time scale.
| Chung, Hoi Sung; Louis, John M; Eaton, William A (2009) Experimental determination of upper bound for transition path times in protein folding from single-molecule photon-by-photon trajectories. Proc Natl Acad Sci U S A 106:11837-44 |
| Best, Robert B; Merchant, Kusai A; Gopich, Irina V et al. (2007) Effect of flexibility and cis residues in single-molecule FRET studies of polyproline. Proc Natl Acad Sci U S A 104:18964-9 |
| Merchant, Kusai A; Best, Robert B; Louis, John M et al. (2007) Characterizing the unfolded states of proteins using single-molecule FRET spectroscopy and molecular simulations. Proc Natl Acad Sci U S A 104:1528-33 |
