title: Characterizing the unfolded states of proteins using single-molecule FRET spectroscopy and molecular simulations.
abstract: To obtain quantitative information on the size and dynamics of unfolded proteins we combined single-molecule lifetime and intensity FRET measurements with molecular simulations. We compared the unfolded states of the 64-residue, alpha/beta protein L and the 66-residue, all-beta cold-shock protein CspTm. The average radius of gyration (Rg) calculated from FRET data on freely diffusing molecules was identical for the two unfolded proteins at guanidinium chloride concentrations >3 M, and the FRET-derived Rg of protein L agreed well with the Rg previously measured by equilibrium small-angle x-ray scattering. As the denaturant concentration was lowered, the mean FRET efficiency of the unfolded subpopulation increased, signaling collapse of the polypeptide chain, with protein L being slightly more compact than CspTm. A decrease in Rg with decreasing denaturant was 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 from either increased interresidue attraction or decreased excluded volume. In contrast to both the FRET and simulation results, previous time-resolved small-angle x-ray scattering experiments showed no collapse for protein L. 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 with the donor life-time of approximately 2 ns, whereas 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.
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA.
authors.forename: Kusai A
authors.initials: KA
authors.lastname: Merchant
authors.forename: Robert B
authors.initials: RB
authors.lastname: Best
authors.forename: John M
authors.initials: JM
authors.lastname: Louis
authors.forename: Irina V
authors.initials: IV
authors.lastname: Gopich
authors.forename: William A
authors.initials: WA
authors.lastname: Eaton
mesh_terms: D019295:Computational Biology / Q000379:methods*; D003198:Computer Simulation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008958:Models, Molecular; D008968:Molecular Conformation; D017785:Photons; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D012984:Software; D014867:Water / Q000737:chemistry; D014965:X-Rays
chemical_list: D014867:Water