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Authordc.contributor.authorZamora, Ricardo A. 
Authordc.contributor.authorFuentes Lemus, Eduardo 
Authordc.contributor.authorBarrias, Pablo 
Authordc.contributor.authorHerrera Morandé, Alejandra 
Authordc.contributor.authorMura, Francisco 
Authordc.contributor.authorGuixé, Victoria 
Authordc.contributor.authorCastro Fernández, Víctor 
Authordc.contributor.authorRojas, Tomás 
Authordc.contributor.authorLópez Alarcón, Camilo 
Authordc.contributor.authorAguirre, Paulina 
Authordc.contributor.authorRivas Aravena, Andrea 
Authordc.contributor.authorAspée, Alexis 
Cita de ítemdc.identifier.citationFree Radical Biology and Medicine 150 (2020) 40–52es_ES
Abstractdc.description.abstractThe enhanced green fluorescent protein (eGFP) is one of the most employed variants of fluorescent proteins. Nonetheless little is known about the oxidative modifications that this protein can undergo in the cellular milieu. The present work explored the consequences of the exposure of eGFP to free radicals derived from γ-radiolysis of water, and AAPH thermolysis. Results demonstrated that protein crosslinking was the major pathway of modification of eGFP towards these oxidants. As evidenced by HPLC-FLD and UPLC-MS, eGFP crosslinking would occur as consequence of a mixture of pathways including the recombination of two protein radicals, as well as secondary reactions between nucleophilic residues (e.g. lysine, Lys) with protein carbonyls. The first mechanism was supported by detection of dityrosine and cysteine-tyrosine bonds, whilst evidence of formation of protein carbonyls, along with Lys consumption, would suggest the formation and participation of Schiff bases in the crosslinking process. Despite of the degree of oxidative modifications elicited by peroxyl radicals (ROO•) generated from the thermolysis of AAPH, and free radicals generated from γ-radiolysis of water, that were evidenced at amino acidic level, only the highest dose of γ-irradiation (10 kGy) triggered significant changes in the secondary structure of eGFP. These results were accompanied by the complete loss of fluorescence arising from the chromophore unit of eGFP in γ-irradiation-treated samples, whereas it was conserved in ROO•-treated samples. These data have potential biological significance, as this fluorescent protein is widely employed to study interactions between cytosolic proteins; consequently, the formation of fluorescent eGFP dimers could act as artifacts in such experiments.es_ES
Patrocinadordc.description.sponsorshipComision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) CONICYT FONDECYT 1190881 1191321 3170590 FONDEQUIP EQM 140151 EQM 120065 DICYT 021941AL_POSTDOC Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) 21160605es_ES
Type of licensedc.rightsAttribution-NonCommercial-NoDerivs 3.0 Chile*
Link to Licensedc.rights.uri*
Sourcedc.sourceFree Radical Biology and Medicinees_ES
Keywordsdc.subjectFluorescent proteinses_ES
Keywordsdc.subjectProtein cross-linkinges_ES
Keywordsdc.subjectCysteine-tyrosine bondses_ES
Keywordsdc.subjectdi-tyrosine bondses_ES
Títulodc.titleFree radicals derived from γ-radiolysis of water and AAPH thermolysis mediate oxidative crosslinking of eGFP involving Tyr-Tyr and Tyr-Cys bonds: the fluorescence of the protein is conserved only towards peroxyl radicalses_ES
Document typedc.typeArtículo de revistaes_ES
dcterms.accessRightsdcterms.accessRightsAcceso Abierto
Indexationuchile.indexArtículo de publicación ISI
Indexationuchile.indexArtículo de publicación SCOPUS

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Attribution-NonCommercial-NoDerivs 3.0 Chile
Except where otherwise noted, this item's license is described as Attribution-NonCommercial-NoDerivs 3.0 Chile