Slow unfolding pathway of hyperthermophilic Tk-RNase H2 examined by pulse proteolysis using the stable protease...

The unfolding speed of some hyperthermophilic proteins is significantly slower than those of their mesostable homologues. Ribonuclease H2 from the hyperthermophilic archaeon Thermococcus kodakarensis (Tk-RNase H2) is stabilized by its remarkably slow unfolding rate. In this work, we examined the slow unfolding pathway of Tk-RNase H2 by pulse proteolysis using a superstable subtilisin-like serine protease from T. kodakarensis (Tk-subtilisin). Tk-subtilisin has enzymatic activity in highly concentrated guanidine hydrochloride (GdnHCl), in which Tk-RNase H2 unfolds slowly. The native state of Tk-RNase H2 was completely resistant to Tk-subtilisin, whereas the unfolded state (induced by 4 M GdnHCl) was degraded by Tk-subtilisin. Degradation products of Tk-RNase H2 created from pulse proteolysis during its unfolding were detected by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We identified the cleavage sites in Tk-RNase H2 by N-terminal sequencing and mass spectrometry and constructed mimics of the unfolding intermediate of Tk-RNase H2 by protein engineering. The mimics were biophysically characterized. We found that the native state of Tk-RNase H2 (N-state) changed to the I(A)-state that was digested by Tk-subtilisin in the early stage of unfolding. In the slow unfolding pathway, the I(A)-state shifted to two intermediate forms, I(B)-state and I(C)-state. The I(B)-state was digested by Tk-subtilisin in the C-terminal region, but the I(C)-state was a Tk-subtilisin resistant form. These states gradually unfolded through the I(D)-state, in which the N-terminal region was digested. The results indicate that pulse proteolysis, by a superstable protease, was a suitable strategy and an effective tool for analyzing intermediate structures of proteins with slow unfolding properties. We also showed that the N-terminal region contributes to the slow unfolding of Tk-RNase H2, and the C-terminal region is important for folding and stability.