1. Direct Observation of the Three-State Folding of a Single Protein Molecule Introduction: -E.Coli Ribonuclease H (Rnase H) -155 residue single-domain protein -It has been suggested that the core of the protein folds first followed by the remaining polypeptide. By applying single-molecule techniques the nature of the Rnase H folding is investigated. Ciro Cecconi, Elizabeth A.Shank, Carlos Bustamante, and Susan Marqusee

2. Setup and results: When RNase H is attached to DNA handles is still seems to be folded correctly and it retains most of its activity

3. Fitting the force-extension curves with the “worm-chain model” indicated that the high-force transition (19 pN) corresponded to complete unfolding. The low-force transition (5.5 pN) only involved a portion of the polypeptide chain. The gap between the stretching and relaxation curves at ~ 5.5 pN supports the idea of partial refolding. The refolding intermediate unfolds again at ~ 5.5 pN in the next stretching cycle, unless the relaxed protein has time to fully refold before being stretched again.

4. This leads to the determination of k (obs)(I→N) = 0.17 ± 0.03 s -1 and Δx ‡ I→N = 1.5 ± 0.3 nm compared to k I→N(bulk) = 0.74 ± 0.02 s -1 The probability of refolding from I to N (P f ) as a function of force (F) and time (t) was fit to Whether the protein had completely refolded into its native structure was monitored as the presence of high-force transition for the subsequent stretching curves Fig. 2

5. The difference between the unfolding (N→U) and the refolding transition suggests that at the rate of pulling the states can not equilibrate High-force unfolding Low-force unfolding (red) and refolding (blue) The unfolding and refolding transitions of the intermediate (I→U and U→I) occur reversibly under the experimental conditions used

6. Pulling the molecule at different loading rates leads to the determination of k (obs)(N→U) = 3 (±2) x 10 -4 s -1 and Δx ‡ N→U = 2.0 ± 0.1 nm compared to k N→U(bulk) = 1.7 (±0.04) x 10 -5 s -1 Fig. 2

7. That the I→U and U→I transition occur reversibly under the experimental settings is consistent with the observation, that the force-extension curves occasionally show rapid fluctuations in extension at ~ 5.5pN

8. When held at a force near 5.5 pN the RNase H molecule displayed bistability. Under these conditions the molecular extension shifted rapidly by ~ 15 nm. Fig. 3 Unfolded state Intermediate state The force-dependent rates of unfolding and refolding were determined from the lifetime of the U and I state seen in fig. 3a. The position of the transition state between I and U was estimated by fitting the rates to this ”Arrhenius-like” equation: This yielded Δx ‡ I→U = 5 ± 1 nm and Δx ‡ U→I = 6 ± 1 nm

9. Fig. 3 The thermodynamics of the U→I transition (ΔG (UI) ) was evaluated with three different methods yielding ΔG (UI) = 4 ± 3 kcal/mol, ΔG (UI) = 4 ± 2 kcal/mol and ΔG (UI) = 3.8 ± 0.8 kcal/mol compared to ΔG (UI)(bulk) = 3.6 ± 0.1 kcal/mol The similarities between the values obtained here and the values from bulk studies suggest that the intermediate detected in this single-molecule study correlates with that sampled in solution

10. To further confirm this relationship similar experiments were performed with a variant of RNase H (I53D) that displays two-state folding in solution Fig. 3 Using this variant no intermediate can be detected

11. Sometimes during the constant force experiments the hopping between the U and I states spontaneously ceased The termination of hopping was preceeded by a contraction. The size of this contraction corresponds well to that expected for the I → N transition at the given force. Stretching the molecule after hopping ceased always resulted in a high-force unfolding transition. Fig. 3 This indicates that the intermediate exists on-pathway to the folded state

12. Finally a representation of the energy landscape (A) could be constructed. By including the calculated distances between the transition states and the different states of the protein ( Δx ‡ values) the free energy reaction profile (B) of RNase H could be estimated

13. Conclusion: The folding process in this study resembles the folding process in solution The large extension-distance between the I and the I →U transition state indicates that the intermediate is a structure that can deform elastically a lot before being committed to unfolding This large distance and the fact that low forces are requried to unfold the structure suggest that the intermediate only forms weak, possibly transient tertiary structures and that it therefore resembles a molten globule The intermediate is on-pathway to the folded state