Effects of changing the interaction between subdomains on the thermostability of Bacillus neutral proteases
| Autor: | Bart Hazes, Gerrit Vriend, B Vandenburg, V.G.H. Eijsink, B Vandervinne, Gerhardus Venema |
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| Přispěvatelé: | Groningen Biomolecular Sciences and Biotechnology |
| Rok vydání: | 1992 |
| Předmět: |
MESENTERICUS STRAIN-76
DNA Bacterial Autolysis (biology) Proteases STABILIZATION Protein Folding Mutant Molecular Sequence Data PROTEIN TRANSITION-STATE SUBTILIS Protein Engineering Biochemistry AUTOLYSIS Bacterial Proteins Structural Biology Thermolysin NUCLEOTIDE-SEQUENCE Sequence Homology Nucleic Acid Enzyme Stability Amino Acid Sequence Thermolabile DISULFIDE BONDS Site-directed mutagenesis Molecular Biology THERMOLYSIN KINETICS Thermostability Base Sequence Sequence Homology Amino Acid Chemistry UNFOLDING Metalloendopeptidases Protein engineering LIMITED PROTEOLYSIS AMINO-ACID SUBDOMAIN Models Chemical Biophysics Mutagenesis Site-Directed Thermodynamics MOLTEN GLOBULE STATE Bacillus subtilis Plasmids |
| Zdroj: | Proteins-Structure Function and Bioinformatics, 14(2), 224-236. Wiley |
| ISSN: | 0887-3585 |
| Popis: | Variants of the thermolabile neutral protease (Npr) of B. subtilis (Npr-sub) and the thermostable neutral protease of B. stearothermophilus (Npr-ste) were produced by means of site-directed mutagenesis and the effects of the mutations on thermostability were determined. Mutations were designed to alter the interaction between the middle and C-terminal subdomain of these enzymes. In all Nprs a cluster of hydrophobic contacts centered around residue 315 contributes to this interaction. In thermostable Nprs (like Npr-ste) a 10 residue beta-hairpin, covering the domain interface, makes an additional contribution. The hydrophobic residue at position 315 was replaced by smaller amino acids. In addition, the beta-hairpin was deleted from Npr-ste and inserted into Npr-sub. The changes in thermostability observed after these mutations confirmed the importance of the hydrophobic cluster and of the beta-hairpin for the structural integrity of Nprs. Combined mutants showed that the effects of individual mutations affecting the interaction between the subdomains were not additive. The effects on thermostability decreased as the strength of the subdomain interaction increased. The results show that once the subdomain interface is sufficiently stabilized, additional stabilizing mutations at the same interface do not further increase thermostability. The results are interpreted on the basis of a model for the thermal inactivation of neutral proteases, in which it is assumed that inactivation results from the occurrence of local unfolding processes that render these enzymes susceptible to autolysis. |
| Databáze: | OpenAIRE |
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