Refolding of the non-specific neutral protease from Bacillus stearothermophilus proceeds via an autoproteolytically sensitive intermediate.

Peter Dürrschmidt, Johanna Mansfeld, Renate Ulbrich-Hofmann

Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, 06099 Halle/Saale, Germany. Peter.Duerrschmidt@wacker.com

Biophysical chemistry 2010 Mar

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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. Copyright 2010 Elsevier B.V. All rights reserved.

Citation

Peter Dürrschmidt, Johanna Mansfeld, Renate Ulbrich-Hofmann. Refolding of the non-specific neutral protease from Bacillus stearothermophilus proceeds via an autoproteolytically sensitive intermediate. Biophysical chemistry. 2010 Mar;147(1-2):66-73