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Analysis of structural determinants of the stability of thermolysin-like proteases by molecular modelling and site-directed mutagenesis

Veltman, OR., Vriend, G., Middelhoven, PJ., vandenBurg, B., Venema, G. & Eijsink, VGH., Dec-1996, In : Protein Engineering. 9, 12, p. 1181-1189 9 p.

Research output: Contribution to journalArticleAcademicpeer-review

  • OR Veltman
  • G Vriend
  • PJ Middelhoven
  • B vandenBurg
  • G Venema
  • VGH Eijsink

The thermolysin-like protease (TLP) produced by Bacillus stearothermophilus CU21 (TLP-ste) differs at 43 positions from the more thermally stable thermolysin (containing 316 residues in total), Of these differences, 26 were analysed by studying the effect of replacing residues in TLP-ste by the corresponding residues in thermolysin, Several stabilizing mutations were identified but, remarkably, considerable destabilizing mutational effects were also found, A Tyr-rich three residue insertion in TLP-ste (the only deletional/insertional difference between the two enzymes) appeared to make an important contribution to the stability of the enzyme. Mutations with large effects on stability were all localized in the beta-pleated N-terminal domain of TLP-ste, confirming;observations that this domain has a lower intrinsic stability than the largely alpha-helical C-terminal domain, Rigidifying mutations such as Gly58 --> Ala and Ala69 --> Pro were among the most stabilizing ones, Apart from this observation, the analyses did not reveal general rules for stabilizing proteins. Instead, the results highlight the importance of context in evaluating the stability effects of mutations.

Original languageEnglish
Pages (from-to)1181-1189
Number of pages9
JournalProtein Engineering
Volume9
Issue number12
Publication statusPublished - Dec-1996

    Keywords

  • autolysis, Bacillus, neutral protease, stability, thermolysin, STEAROTHERMOPHILUS NEUTRAL PROTEASE, BACILLUS-STEAROTHERMOPHILUS, THERMAL-STABILITY, NUCLEOTIDE-SEQUENCE, CRYSTAL-STRUCTURE, TRANSITION-STATE, THERMOSTABILITY, CLONING, GENE, PROTEINS

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