The molten-globule residual structure is critical for reflavination of glucose oxidase

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388971%3A_____%2F17%3A00484183" target="_blank" >RIV/61388971:_____/17:00484183 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/61989592:15310/17:73586491

Výsledek na webu

<a href="http://dx.doi.org/10.1016/j.bpc.2017.08.009" target="_blank" >http://dx.doi.org/10.1016/j.bpc.2017.08.009</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.bpc.2017.08.009" target="_blank" >10.1016/j.bpc.2017.08.009</a>

Jazyk výsledku

angličtina

Název v původním jazyce

The molten-globule residual structure is critical for reflavination of glucose oxidase

Popis výsledku v původním jazyce

Glucose oxidase (GOX) is a homodimeric glycoprotein with tightly bound one molecule of FAD cofactor per monomer of the protein. GOX has numerous applications, but the preparation of biotechnologically interesting GOX sensors requires a removal of the native FAD cofactor. This process often leads to unwanted irreversible deflavination and, as a consequence, to the low enzyme recovery. Molecular mechanisms of reversible reflavination are poorly understood, our current knowledge is based only on empiric rules, which is clearly insufficient for further development. To develop conceptual understanding of flavin-binding competent states, we studied the effect of deflavination protocols on conformational properties of GOX. After deflavination, the apoform assembles into soluble oligomers with nearly native-like holoform secondary structure but largely destabilized tertiary structure presumambly due to the packing density defects around the vacant flavin binding site. The reflavination is cooperative but not fully efficient, after the binding the flavin cofactor, the protein directly disassembles into native homodimers while the fraction of oligomers remains irreversibly inactivated. Importantly, the effect of Hofineister salts on the conformational properties of GOX and reflavination efficiency indicates that the native-like residual tertiary structure in the molten-globule states favorably supports the reflavination and minimizes the inactivated oligomers. We interpret our results by combining the ligand-induced changes in quaternary structure with salt-sensitive, non-equilibrated conformational selection model. In summary, our work provides the very first steps toward molecular understanding the complexity of the GOX reflavination mechanism.

Název v anglickém jazyce

The molten-globule residual structure is critical for reflavination of glucose oxidase

Popis výsledku anglicky

Glucose oxidase (GOX) is a homodimeric glycoprotein with tightly bound one molecule of FAD cofactor per monomer of the protein. GOX has numerous applications, but the preparation of biotechnologically interesting GOX sensors requires a removal of the native FAD cofactor. This process often leads to unwanted irreversible deflavination and, as a consequence, to the low enzyme recovery. Molecular mechanisms of reversible reflavination are poorly understood, our current knowledge is based only on empiric rules, which is clearly insufficient for further development. To develop conceptual understanding of flavin-binding competent states, we studied the effect of deflavination protocols on conformational properties of GOX. After deflavination, the apoform assembles into soluble oligomers with nearly native-like holoform secondary structure but largely destabilized tertiary structure presumambly due to the packing density defects around the vacant flavin binding site. The reflavination is cooperative but not fully efficient, after the binding the flavin cofactor, the protein directly disassembles into native homodimers while the fraction of oligomers remains irreversibly inactivated. Importantly, the effect of Hofineister salts on the conformational properties of GOX and reflavination efficiency indicates that the native-like residual tertiary structure in the molten-globule states favorably supports the reflavination and minimizes the inactivated oligomers. We interpret our results by combining the ligand-induced changes in quaternary structure with salt-sensitive, non-equilibrated conformational selection model. In summary, our work provides the very first steps toward molecular understanding the complexity of the GOX reflavination mechanism.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10606 - Microbiology

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2017

Kód důvěrnosti údajů

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

Název periodika

Biophysical Chemistry

ISSN

0301-4622

e-ISSN

—

Svazek periodika

230

Číslo periodika v rámci svazku

NOV 2017

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

10

Strana od-do

74-83

Kód UT WoS článku

000413390200010

EID výsledku v databázi Scopus

2-s2.0-85028733236