title: Fast Folding Dynamics of an Intermediate State in RNase H Measured by Single-Molecule FRET.
abstract: We have studied the folding kinetics of the core intermediate (I) state of RNase H by using a combination of single-molecule FRET (smFRET) and hidden Markov model analysis. To measure fast dynamics in thermal equilibrium as a function of the concentration of the denaturant GdmCl, a special FRET labeled variant, RNase H 60-113, which is sensitive to folding of the protein core, was immobilized on PEGylated surfaces. Conformational transitions between the unfolded (U) state and the I state could be described by a two-state model within our experimental time resolution, with millisecond mean residence times. The I state population was always a minority species in the entire accessible range of denaturant concentrations. By introducing the measured free energy differences between the U and I states as constraints in global fits of the GdmCl dependence of FRET histograms of a differently labeled RNase H variant (RNase H 3-135), we were able to reveal the free energy differences and, thus, population ratios of all three macroscopic state ensembles, U, I and F (folded state) as a function of denaturant concentration.
authors.affiliation: Institute of Applied Physics, Karlsruhe Institute of Technology (KIT) , Wolfgang-Gaede-Strasse 1, 76131 Karlsruhe, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Stockmar
authors.affiliation: Institute of Applied Physics, Karlsruhe Institute of Technology (KIT) , Wolfgang-Gaede-Strasse 1, 76131 Karlsruhe, Germany.
authors.forename: Andrei Yu
authors.initials: AY
authors.lastname: Kobitski
authors.affiliation: Institute of Applied Physics, Karlsruhe Institute of Technology (KIT) , Wolfgang-Gaede-Strasse 1, 76131 Karlsruhe, Germany.|Institute of Nanotechnology, Karlsruhe Institute of Technology (KIT) , Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.|Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT) , Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.|Department of Physics, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Gerd Ulrich
authors.initials: GU
authors.lastname: Nienhaus
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D017510:Protein Folding; D016914:Ribonuclease H / Q000737:chemistry*
chemical_list: D016914:Ribonuclease H