title: Refolding of the non-specific neutral protease from Bacillus stearothermophilus proceeds via an autoproteolytically sensitive intermediate.
abstract: A very thermostable variant of the thermolysin-like protease from Bacillus stearothermophilus (G8C/N60C) was previously created by introduction of a disulfide bond into the cysteine-free pseudo-wild type variant (pWT) and thus fixing the unfolding region 56-69. In the present paper, we show that G8C/N60C and pWT can be reactivated from the completely unfolded states, accessible at >or=7.5M guanidine hydrochloride, and analyze the kinetics of folding, autoproteolytic degradation and aggregation. From changes in the fluorescence spectra with time of renaturation, it can be concluded that a folding intermediate with native-like structure, but which is still inactive and sensitive to autoproteolysis, is rapidly formed after renaturation has initiated. The critical region 56-69 of pWT is involved in the autoproteolytic sensitivity of the intermediate as we conclude from the differences in the chevron plots of the first-order rate constants of reactivation and the fragmentation patterns in SDS-PAGE of pWT and G8C/N60C.
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, 06099 Halle/Saale, Germany. Peter.Duerrschmidt@wacker.com
authors.forename: Peter
authors.initials: P
authors.lastname: Dürrschmidt
authors.forename: Johanna
authors.initials: J
authors.lastname: Mansfeld
authors.forename: Renate
authors.initials: R
authors.lastname: Ulbrich-Hofmann
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D008027:Light; D008958:Models, Molecular; D017510:Protein Folding; D020673:Protein Renaturation; D020836:Protein Structure, Quaternary; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D010450:Endopeptidases; C065625:neutral protease, Bacillus stearothermophilus