Journal article
Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease
University of Szeged1
Czech Academy of Sciences2
University of Copenhagen3
Department of Chemistry, Technical University of Denmark4
Metalloprotein Chemistry and Engineering, Department of Chemistry, Technical University of Denmark5
University of Tsukuba6
MTA-SZTE Bioinorganic Chemistry Research Group7
The nuclease domain of colicin E7 (NColE7) cleaves DNA nonspecifically. The active center is a Zn(2+)-containing HNH motif at the C-terminus. The N-terminal loop is essential for the catalytic activity providing opportunity for allosteric modulation of the enzyme. To identify the key residues responsible for the structural integrity of NColE7, a virtual alanine scan was performed on a semiempirical quantum chemical level within the 25 residue long N-terminal sequence (446-470).
Based on the calculations the T454A/K458A/W464A-NColE7 triple mutant (TKW) was expressed and purified. According to the agarose gel electrophoresis experiments and linear dichroism spectra the catalytic activity of the TKW mutant decreased in comparison with wild-type NColE7. The distorted structure and weakened Zn(2+) binding may account for this as revealed by circular dichroism spectra, mass spectrometry, fluorescence-based thermal analysis and isothermal microcalorimetric titrations.
Remarkably, the substrate induced the folding of the mutant protein.
Language: | English |
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Publisher: | Springer Berlin Heidelberg |
Year: | 2014 |
Pages: | 1295-1303 |
ISSN: | 14321327 and 09498257 |
Types: | Journal article |
DOI: | 10.1007/s00775-014-1186-6 |
ORCIDs: | Christensen, Hans Erik Mølager and 0000-0002-9241-4352 |
Binding affinity Calorimetry HASH(0x482a968) Protein engineering Substrate induced folding Zinc nuclease
Alanine Amino Acid Sequence Binding Sites Biochemistry, general Biomedical and Life Sciences ColE7 protein, E coli Colicins DNA Life Sciences Microbiology Models, Molecular Molecular Sequence Data Mutant Proteins Mutation Protein Binding Protein Conformation Protein Engineering Protein Folding Zinc