Cumulative stabilizing effects of hydrophobic interactions on the surface of the neutral protease from Bacillus subtilis

Francesco Frigerio, Immaculada Margarit, Renzo Nogarotto, Vincenzo De Filippis, Guido Grandi

Research output: Contribution to journalArticle

Abstract

Using genetically engineered mutants of the neutral protease from Bacillus stearothermophilus (BsteNP), it had been shown that the surface-exposed structural moth constituted by Phe63 embedded in a four amino acid hydrophobic pocket is critical for the thermal stability of the thermophilic neutral proteases from Bacilli. To measure the stabilizing contribution of each hydrophobic interaction taking place between Phe63 and the hydrophobic pocket, we grafted this structural motif in the neutral protease from the mesophile Bacillus subtilis (BsubNP). This was accomplished by first creating the Thr63→Phe mutant of BsubNP and then generating a series of mutants in which the four amino acids which in thermolysin surround Phe63 and form the hydrophobic pocket were added one after the other. By analysing the thermal stability of each mutant it was found that the 2°C destabilizing effect of the Thr63→Phe substitution was completely suppressed by the addition of the four amino acid hydrophobic pocket, each replacement providing a stabilizing contribution of approximately 0.8-1°C. These results are discussed in the light of the peculiar mechanism of thermal inactivation of proteolytic enzymes.

Original languageEnglish
Pages (from-to)439-445
Number of pages7
JournalProtein Engineering
Volume9
Issue number5
Publication statusPublished - May 1996

Keywords

  • Bacillus subtilis
  • Hydrophobic interactions
  • Neutral proteases
  • Protein stability

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Frigerio, F., Margarit, I., Nogarotto, R., De Filippis, V., & Grandi, G. (1996). Cumulative stabilizing effects of hydrophobic interactions on the surface of the neutral protease from Bacillus subtilis. Protein Engineering, 9(5), 439-445.