Single-molecule Förster resonance energy transfer study of protein dynamics under denaturing conditions
- 12 October 2005
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 102 (43), 15471-15476
- https://doi.org/10.1073/pnas.0507728102
Abstract
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 ≈20 μs 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 ≈2 s. These data were analyzed to obtain information on the free energy landscape of RNase H in the presence of chemical denaturants.Keywords
This publication has 47 references indexed in Scilit:
- Biocompatible Surfaces for Specific Tethering of Individual Protein MoleculesThe Journal of Physical Chemistry B, 2004
- Comparison of the folding processes of T. thermophilus and E. coli Ribonucleases HJournal of Molecular Biology, 2002
- Persistence of Native-Like Topology in a Denatured Protein in 8 M UreaScience, 2001
- A statistical appraisal of native state hydrogen exchange data: evidence for a burst phase continuum?Journal of Molecular Biology, 2000
- The cooperativity of burst phase reactions exploredJournal of Molecular Biology, 1999
- FAST EVENTS IN PROTEIN FOLDING: The Time Evolution of Primary ProcessesAnnual Review of Physical Chemistry, 1998
- THEORY OF PROTEIN FOLDING: The Energy Landscape PerspectiveAnnual Review of Physical Chemistry, 1997
- Viscosity Dependence of the Folding Rates of ProteinsPhysical Review Letters, 1997
- Folding Pathway of Escherichia coli Ribonuclease HI: A Circular Dichroism, Fluorescence, and NMR StudyBiochemistry, 1995
- Principles that Govern the Folding of Protein ChainsScience, 1973