Direct Observation of the Three-State Folding of a Single Protein Molecule

We used force-measuring optical tweezers to induce complete mechanical unfolding and refolding of individual Escherichia coli ribonuclease H (RNase H) molecules. The protein unfolds in a two-state manner and refolds through an intermediate that correlates with the transient molten globule-like inter...

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Bibliographic Details
Published in:Science (American Association for the Advancement of Science) 2005-09, Vol.309 (5743), p.2057-2060
Main Authors: Cecconi, Ciro, Shank, Elizabeth A, Bustamante, Carlos, Marqusee, Susan
Format: Article
Language:English
Subjects:
Analysis
Biochemistry
Biological and medical sciences
Circular Dichroism
Deoxyribonucleic acid
DNA
DNA - chemistry
Escherichia coli Proteins - chemistry
Fundamental and applied biological sciences. Psychology
Globulins
Kinetics
Least squares
Loading rate
Molecular biology
Molecular biophysics
Molecules
Observation
Optics and Photonics
Protein Folding
Protein refolding
Protein research
Proteins
Ribonuclease H - chemistry
Structure in molecular biology
Thermodynamics
Trajectories
Tridimensional structure
Tweezers
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Summary:We used force-measuring optical tweezers to induce complete mechanical unfolding and refolding of individual Escherichia coli ribonuclease H (RNase H) molecules. The protein unfolds in a two-state manner and refolds through an intermediate that correlates with the transient molten globule-like intermediate observed in bulk studies. This intermediate displays unusual mechanical compliance and unfolds at substantially lower forces than the native state. In a narrow range of forces, the molecule hops between the unfolded and intermediate states in real time. Occasionally, hopping was observed to stop as the molecule crossed the folding barrier directly from the intermediate, demonstrating that the intermediate is on-pathway. These studies allow us to map the energy landscape of RNase H.
ISSN:0036-8075
1095-9203
DOI:10.1126/science.1116702