Počet záznamů: 1
Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease
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SYSNO ASEP 0436908 Druh ASEP J - Článek v odborném periodiku Zařazení RIV J - Článek v odborném periodiku Poddruh J Článek ve WOS Název Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease Tvůrce(i) 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)Celkový počet autorů 8 Zdroj.dok. Journal of Biological Inorganic Chemistry. - : Springer - ISSN 0949-8257
Roč. 19, č. 8 (2014), s. 1295-1303Poč.str. 9 s. Jazyk dok. eng - angličtina Země vyd. DE - Německo Klíč. slova binding affinity ; calorimetry ; zinc nuclease ; substrate induced folding ; protein engineering Vědní obor RIV CE - Biochemie Institucionální podpora UOCHB-X - RVO:61388963 UT WOS 000345403900005 EID SCOPUS 84904753004 DOI 10.1007/s00775-014-1186-6 Anotace 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. Pracoviště Ústav organické chemie a biochemie Kontakt asep@uochb.cas.cz ; Kateřina Šperková, Tel.: 232 002 584 ; Viktorie Chládková, Tel.: 232 002 434 Rok sběru 2015
Počet záznamů: 1