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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388963%3A_____%2F14%3A00436908" target="_blank" >RIV/61388963:_____/14:00436908 - isvavai.cz</a>

Výsledek na webu

<a href="http://dx.doi.org/10.1007/s00775-014-1186-6" target="_blank" >http://dx.doi.org/10.1007/s00775-014-1186-6</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1007/s00775-014-1186-6" target="_blank" >10.1007/s00775-014-1186-6</a>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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 theenzyme. 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. Thedistorted 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 in

Název v anglickém jazyce

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

Popis výsledku anglicky

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 theenzyme. 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. Thedistorted 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 in

Druh

J_x - Nezařazeno - Článek v odborném periodiku (Jimp, Jsc a Jost)

CEP obor

CE - Biochemie

OECD FORD obor

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Projekt

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Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2014

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Journal of Biological Inorganic Chemistry

ISSN

0949-8257

e-ISSN

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Svazek periodika

19

Číslo periodika v rámci svazku

8

Stát vydavatele periodika

DE - Spolková republika Německo

Počet stran výsledku

9

Strana od-do

1295-1303

Kód UT WoS článku

000345403900005

EID výsledku v databázi Scopus

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