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 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.
Cecconi
Ciro
C
Department of Molecular and Cell Biology and Institute for Quantitative Biology.
Shank
Elizabeth A
EA
Bustamante
Carlos
C
Marqusee
Susan
S
D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry*; D055095:Optics and Photonics; D017510:Protein Folding; D016914:Ribonuclease H / Q000737:chemistry*; D013816:Thermodynamics
D029968:Escherichia coli Proteins; D004247:DNA; D016914:Ribonuclease H; C078856:ribonuclease HI