Počet záznamů: 1  

Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease

  1. 1. 0436908 - UOCHB-X 2015 RIV DE eng J - Článek v odborném periodiku
    Németh, E. - Körtvélyesi, T. - Kožíšek, Milan - Thulstrup, P. W. - Christensen, H. E. M. - Asaka, M. N. - Nagata, K. - Gyurcsik, B.
    Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease.
    Journal of Biological Inorganic Chemistry. Roč. 19, č. 8 (2014), s. 1295-1303 ISSN 0949-8257
    Grant ostatní:OPPC(XE) CZ.2.16/3.1.00/24016; Seventh Framework Programme of the European Union(XE) FP7-312284
    Institucionální podpora: RVO:61388963
    Klíčová slova: binding affinity * calorimetry * zinc nuclease * substrate induced folding * protein engineering
    Kód oboru RIV: CE - Biochemie
    Impakt faktor: 2.538, rok: 2014

    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.
    Trvalý link: http://hdl.handle.net/11104/0240863