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Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease
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SYSNO ASEP 0436908 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease Author(s) Németh, E. (HU)
Körtvélyesi, T. (HU)
Kožíšek, Milan (UOCHB-X) RID, ORCID
Thulstrup, P. W. (DK)
Christensen, H. E. M. (DK)
Asaka, M. N. (JP)
Nagata, K. (JP)
Gyurcsik, B. (HU)Number of authors 8 Source Title Journal of Biological Inorganic Chemistry. - : Springer - ISSN 0949-8257
Roč. 19, č. 8 (2014), s. 1295-1303Number of pages 9 s. Language eng - English Country DE - Germany Keywords binding affinity ; calorimetry ; zinc nuclease ; substrate induced folding ; protein engineering Subject RIV CE - Biochemistry Institutional support UOCHB-X - RVO:61388963 UT WOS 000345403900005 EID SCOPUS 84904753004 DOI 10.1007/s00775-014-1186-6 Annotation The nuclease domain of colicin E7 (NColE7) cleaves DNA nonspecifically. The active center is a Zn2+-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 Zn2+ 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. Workplace Institute of Organic Chemistry and Biochemistry Contact asep@uochb.cas.cz ; Kateřina Šperková, Tel.: 232 002 584 ; Viktorie Chládková, Tel.: 232 002 434 Year of Publishing 2015
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