To elucidate the energetic features of the anomalously high-level stabilization of a hyperthermophile pyrrolidone carboxyl peptidase (PfPCP) from a hyperthermophilic archaeon, Pyrococcus furiosus, equilibrium and kinetic studies of the guanidine hydrochloride (GuHCl)-induced unfolding and refolding were carried out with CD measurements at 220 nm in comparison with those from the mesophile homologue (BaPCP) from Bacillus amyloliquefaciens. The mutant protein of PfPCP substituted with Ser at both Cys142 and Cys188 (PfC142/188S) was used. The GuHCl unfolding for PfC142/188S and BaPCP was reversible. It was difficult to obtain the equilibrated unfolding curve of the hyperthermophile proteins at temperatures below 50 degreesC and pH 7, because of the remarkably slow rate of the unfolding. The unfolding for PfC142/188S attained equilibrium after 7 days at 60 degreesC, resulting in the coincidence between the unfolding and refolding curves. The Gibbs energy change of unfolding, DeltaGH2O (56.6 kJ/mol), for PfC142/188S at 60 degreesC and pH 7 was dramatically higher than that (7.6 kJ/mol) for BaPCP at 40 degreesC and pH 7. The unfolding and refolding kinetics for PfC142/188S and BaPCP at both 25 and 60 degreesC at pH 7 were approximated as a single exponential. The rate constant in water (kuH2O) of the unfolding reaction for PfC142/188S (1.6 x 10(-)15 s-1) at 25 degreesC and pH 7 was drastically reduced by 7 orders of magnitude compared to that (1.5 x 10(-)8 s-1) for BaPCP, whereas the refolding rates (krH2O) in water for PfC142/188S (9.3 x 10(-)2 s-1) and BaPCP (3.6 x 10(-)1 s-1) at 25 degreesC and pH 7 were similar. These results indicate that the greater stability of the hyperthermophile PCP was characterized by the drastically slow unfolding rate. Study holds ProTherm entries: 9482, 9483, 9484 Extra Details: additive : EDTA(2 mM),DTE(0.1mM) was added in the experiment hyperthermophilic archaeon; mesophile homologue; Gibbs energy,change of unfolding
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:38 p.m.
|Number of data points||9|
|Proteins||Pyrrolidone-carboxylate peptidase ; Pyrrolidone-carboxylate peptidase|
|Assays/Quantities/Protocols||Experimental Assay: Cm temp:40.0 C ; Experimental Assay: m temp:40.0 C ; Experimental Assay: dG_H2O temp:40.0 C ; Experimental Assay: Cm details:Additives , temp:60.0 C ; Experimental Assay: m details:Additives , temp:60.0 C ; Experimental Assay: dG_H2O details:Additives , temp:60.0 C ; Experimental Assay: Cm temp:60.0 C ; Experimental Assay: m temp:60.0 C ; Experimental Assay: dG_H2O temp:60.0 C|
|Libraries||Mutations for sequence MKVLVTGFEPFGGEKINPTERIAKDLDGIKIGDAQVFGRVLPVVFGKAKEVLEKTLEEIKPDIAIHVGLAPGRSAISIERIAVNAIDARIPDNEGKKIEDEPIVPGAPTAYFSTLPIKKIMKKLHERGIPAYISNSAGLYLCNYVMYLSLHHSATKGYPKMSGFIHVPYIPEQIIDKIGKGQVPPSMCYEMELEAVKVAIEVALEELL|