title: Slow unfolding pathway of hyperthermophilic Tk-RNase H2 examined by pulse proteolysis using the stable protease Tk-subtilisin.
abstract: 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.
authors.affiliation: Department of Material and Life Science, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Okada
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Koga
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Takano
authors.forename: Shigenori
authors.initials: S
authors.lastname: Kanaya
mesh_terms: D002942:Circular Dichroism; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D058849:Protein Refolding; D058767:Protein Unfolding / Q000187:drug effects; D059748:Proteolysis; D016914:Ribonuclease H / Q000737:chemistry*; D013381:Subtilisins / Q000378:metabolism; D019712:Thermococcus / Q000378:metabolism
chemical_list: C091139:ribonuclease HII; D016914:Ribonuclease H; D013381:Subtilisins; D019791:Guanidine