KIT | KIT-Bibliothek | Impressum | Datenschutz

Evidence of a Folding Intermediate in RNase H from Single Molecule FRET Experiments

Rieger, R. 1,2; Kobitski, A. 1,2; Sielaff, H. 1,2; Nienhaus, G. U. ORCID iD icon 1,2
1 Institut für Angewandte Physik (APH), Karlsruher Institut für Technologie (KIT)
2 Center for Functional Nanostructures (CFN), Karlsruher Institut für Technologie (KIT)

Abstract:

Single-molecule Förster resonance energy transfer (FRET) experiments were performed on the enzyme RNase H specifically labeled with a FRET dye pair and diffusing freely in solutions containing between 0 and 6 M of the chemical denaturant GdmCl. We measured FRET efficiency histograms with high statistical accuracy to identify the well-known folding intermediate of RNase H, which escaped observation in our previous smFRET studies on immobilized preparations. Even with excellent data statistics, a folding intermediate is not obvious from the raw data. However, it can be uncovered by a global fitting procedure applied to the FRET histograms at all 22 GdmCl concentrations, in which a number of parameters were constrained. Most importantly, the fractional populations of the folded, unfolded and intermediate states were coupled by assuming the Boltzmann relation and a linear dependence of the free energies on the GdmCl concentration. The analysis not only resolves the apparent discrepancy with other data on RNase H, but yields free energy differences between the three populations in agreement with literature data. In addition, it removes the strong and unexplained broadening of the unfolded-state distribution in the transition region that was seen earlier in the two-state analysis.


Originalveröffentlichung
DOI: 10.1002/cphc.201000693
Scopus
Zitationen: 19
Web of Science
Zitationen: 18
Dimensions
Zitationen: 20
Zugehörige Institution(en) am KIT Institut für Angewandte Physik (APH)
Publikationstyp Zeitschriftenaufsatz
Publikationsjahr 2011
Sprache Englisch
Identifikator ISSN: 1439-4235
KITopen-ID: 1000027127
Erschienen in ChemPhysChem
Verlag John Wiley and Sons
Band 12
Heft 3
Seiten 627-633
Nachgewiesen in Web of Science
Dimensions
Scopus
KIT – Die Forschungsuniversität in der Helmholtz-Gemeinschaft
KITopen Landing Page