Mutational analysis of the thermostable arginine repressor from Bacillus stearothermophilus: dissecting residues involved in DNA binding properties
| Autor: | Vehary Sakanyan, Gregory D. Van Duyne, Nicolas Glansdorff, Pierre Weigel, Jean-Noël Hallet, Masayuki Takahashi, Alain Versavaud, Daniel Charlier, Cécile Fort, Iovka Miltcheva Karaivanova |
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| Přispěvatelé: | Biology, Vrije Universiteit Brussel |
| Rok vydání: | 1999 |
| Předmět: |
DNA
Bacterial Models Molecular Arginine Protein Conformation Mutant Molecular Sequence Data Repressor Biology Conserved sequence Geobacillus stearothermophilus Protein structure Bacterial Proteins Drug Stability Structural Biology Escherichia coli Point Mutation Amino Acid Sequence Molecular Biology Peptide sequence Derepression Conserved Sequence DNA Primers chemistry.chemical_classification Binding Sites Base Sequence Sequence Homology Amino Acid Circular Dichroism Escherichia coli Proteins Temperature Recombinant Proteins Amino acid Repressor Proteins chemistry Biochemistry Mutagenesis Site-Directed |
| Zdroj: | Vrije Universiteit Brussel |
| ISSN: | 0022-2836 |
| Popis: | Recently the crystal structure of the DNA-unbound form of the full-length hexameric Bacillus stearothermophilus arginine repressor (ArgR) has been resolved, providing a possible explanation for the mechanism of arginine-mediated repressor-operator DNA recognition. In this study we tested some of these functional predictions by performing site-directed mutagenesis of distinct amino acid residues located in two regions, the N-terminal DNA-binding domain and the C-terminal oligomerization domain of ArgR. A total of 15 mutants were probed for their capacity to repress the expression of the reporter argC - lacZ gene fusion in Escherichia coli cells. Substitutions of highly conserved amino acid residues in the α2 and α3 helices, located in the winged helix-turn-helix DNA-binding motif, reduced repression. Loss of DNA-binding capacity was confirmed in vitro for the Ser42Pro mutant which showed the most pronounced effect in vivo . In E. coli , the wild-type B. stearothermophilus ArgR molecule behaves as a super-repressor, since recombinant E. coli host cells bearing B. stearothermophilus argR on a multicopy vector did not grow in selective minimal medium devoid of arginine and grew, albeit weakly, when l -arginine was supplied. All mutants affected in the DNA-binding domain lost this super-repressor behaviour. Replacements of conserved leucine residues at positions 87 and/or 94 in the C-terminal domain by other hydrophobic amino acid residues proved neutral or caused either derepression or stronger super-repression. Substitution of Leu87 by phenylalanine was found to increase the DNA-binding affinity and the protein solubility in the context of a double Leu87Phe/Leu94Val mutant. Structural modifications occasioned by the various amino acid substitutions were confirmed by circular dichroism analysis and structure modelling. |
| Databáze: | OpenAIRE |
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