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Refolding of the non-specific neutral protease fromBacillus stearothermophilusproceeds via an autoproteolytically sensitive intermediate

Biophysical Chemistry
Publication Date
DOI: 10.1016/j.bpc.2010.01.001
  • Autoproteolysis
  • Disulfide Bond
  • Folding Intermediate
  • Neutral Protease
  • Reactivation
  • Refolding


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 ≥ 7.5 M 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.

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