Strengthening the dimerisation interface of Lac repressor increases its thermostability by 40 deg. C.
We increased drastically the heat stability of Lac repressor (LacR) of Escherichia coli. Wild-type tetrameric LacR denatures irreversibly at 53 degrees C. Improving hydrophobic packing at the dimerisation interface by a single substitution increases LacR heat-resistance by 40 deg. C without abolishing inducer binding at high and low temperatures. Tetrameric LacR mutants carrying substitutions of the positively charged amino acid Lys84 by each of the hydrophobic amino acids Leu, Ile and Met resist heating to temperatures up to 93 degrees C. We performed IPTG binding assays at 80 degrees C and found the mutant Lac repressors active and, thus, the core intact. Furthermore, the activity of LacR following heating is shown at room temperature by a gel retardation assay, which demonstrates normal oligomerisation state and function of the headpiece. The same mutations (K84L/I/M) in the dimer LacR331stop, carrying a stop codon in amino acid 331, increase thermostability of the dimer from 47 degrees C to 87 degrees C. LacRK84M represses beta-galactosidase activity in vivo as well as the wild-type and is sufficiently induced to allow growth on lactose. The results with both tetramer and dimer variants of LacR indicate mutual stabilisation of the tetramerisation region and the stable core.
Gerk
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LP
Institut für Genetik, Universität zu Köln, Köln, Weyertal 121, 50931, Germany. Lily.pereg.gerk.com
Leven
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O
Müller-Hill
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B
D000494:Allosteric Regulation; D019943:Amino Acid Substitution / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D018388:Codon, Terminator / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D007763:Lac Operon / Q000235:genetics; D056968:Lac Repressors; D007785:Lactose / Q000378:metabolism; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009875:Operator Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics
D001426:Bacterial Proteins; D018388:Codon, Terminator; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D012097:Repressor Proteins; D007544:Isopropyl Thiogalactoside; D007785:Lactose; D008239:Lysine