Single-molecule Forster resonance energy transfer study of protein dynamics under denaturing conditions.
Proteins are highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of denaturants, the heterogeneity is greatly enhanced, and fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways. Here, we have studied the structure and dynamics of the small enzyme ribonuclease HI (RNase H) in the presence of the chemical denaturant guanidinium chloride (GdmCl) using single-molecule fluorescence microscopy, with a particular focus on the characterization of the unfolded-state ensemble. A dye pair was specifically attached to the enzyme to measure structural changes through Förster resonance energy transfer (FRET). Enzyme immobilization on star-polymer surfaces that were specially developed for negligible interaction with folded and unfolded proteins enabled us to monitor conformational changes of individual proteins for several hundred seconds. FRET efficiency histograms were calculated from confocal scan images. They showed an expansion of the unfolded proteins with increasing GdmCl concentration. Cross-correlation analysis of donor and acceptor fluorescence intensity time traces from single molecules revealed reconfiguration of the polypeptide chain on a timescale of approximately equal to 20 micros at 1.7 M GdmCl. Slow conformational dynamics gave rise to characteristic, stepwise FRET efficiency changes. Transitions between folded and unfolded enzyme molecules occurred on the 100-s timescale, in excellent agreement with bulk denaturation experiments. Transitions between unfolded conformations were more frequent, with characteristic times of approximately equal to 2 s. These data were analyzed to obtain information on the free energy landscape of RNase H in the presence of chemical denaturants.
Kuzmenkina
Elza V
EV
Department of Biophysics, University of Ulm, Albert Einstein Allee 11, 89081 Ulm, Germany.
Heyes
Colin D
CD
Nienhaus
G Ulrich
GU
D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D011489:Protein Denaturation; D017510:Protein Folding; D016914:Ribonuclease H / Q000737:chemistry*; D013997:Time Factors
D016914:Ribonuclease H