The role of local and remote amino acid substitutions for optimizing fluorescence in bacteriophytochromes: A case study on iRFP

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dc.contributor.authorBuhrke, David
dc.contributor.authorVélazquez Escobar, Francisco
dc.contributor.authorSauthof, Luisa
dc.contributor.authorWilkening, Svea
dc.contributor.authorHerder, Nico
dc.contributor.authorTavraz, Neslihan N.
dc.contributor.authorWilloweit, Mario
dc.contributor.authorKeidel, Anke
dc.contributor.authorUtesch, Tillmann
dc.contributor.authorMroginski, Maria-Andrea
dc.contributor.authorSchmitt, Franz-Josef
dc.contributor.authorHildebrandt, Peter
dc.contributor.authorFriedrich, Thomas
dc.date.accessioned2019-07-05T11:56:55Z
dc.date.available2019-07-05T11:56:55Z
dc.date.issued2016-06-22
dc.description.abstractBacteriophytochromes are promising tools for tissue microscopy and imaging due to their fluorescence in the near-infrared region. These applications require optimization of the originally low fluorescence quantum yields via genetic engineering. Factors that favour fluorescence over other non-radiative excited state decay channels are yet poorly understood. In this work we employed resonance Raman and fluorescence spectroscopy to analyse the consequences of multiple amino acid substitutions on fluorescence of the iRFP713 benchmark protein. Two groups of mutations distinguishing iRFP from its precursor, the PAS-GAF domain of the bacteriophytochrome P2 from Rhodopseudomonas palustris, have qualitatively different effects on the biliverdin cofactor, which exists in a fluorescent (state II) and a non-fluorescent conformer (state I). Substitution of three critical amino acids in the chromophore binding pocket increases the intrinsic fluorescence quantum yield of state II from 1.7 to 5.0% due to slight structural changes of the tetrapyrrole chromophore. Whereas these changes are accompanied by an enrichment of state II from ~40 to ~50%, a major shift to ~88% is achieved by remote amino acid substitutions. Additionally, an increase of the intrinsic fluorescence quantum yield of this conformer by ~34% is achieved. The present results have important implications for future design strategies of biofluorophores.en
dc.description.sponsorshipDFG, 221545957, SFB 1078: Proteinfunktion durch Protonierungsdynamiken
dc.description.sponsorshipDFG, 53182490, EXC 314: Unifying Concepts in Catalysisen
dc.identifier.eissn2045-2322
dc.identifier.urihttps://depositonce.tu-berlin.de/handle/11303/9595
dc.identifier.urihttp://dx.doi.org/10.14279/depositonce-8640
dc.language.isoenen
dc.relation.ispartof10.14279/depositonce-8309
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/en
dc.subject.ddc540 Chemie und zugeordnete Wissenschaftenen
dc.subject.otheramino acid substitutionen
dc.subject.otheriRFPen
dc.subject.otherbacteriophytochromesen
dc.subject.otherproteinen
dc.subject.otherfluorescenceen
dc.titleThe role of local and remote amino acid substitutions for optimizing fluorescence in bacteriophytochromes: A case study on iRFPen
dc.typeArticleen
dc.type.versionpublishedVersionen
dcterms.bibliographicCitation.articlenumber28444en
dcterms.bibliographicCitation.doi10.1038/srep28444en
dcterms.bibliographicCitation.journaltitleScientific Reportsen
dcterms.bibliographicCitation.originalpublishernameNature Publishing Groupen
dcterms.bibliographicCitation.originalpublisherplaceLondonen
dcterms.bibliographicCitation.volume6en
tub.accessrights.dnbfreeen
tub.affiliationFak. 2 Mathematik und Naturwissenschaften::Inst. Chemie::FG Bioenergetikde
tub.affiliation.facultyFak. 2 Mathematik und Naturwissenschaftende
tub.affiliation.groupFG Bioenergetikde
tub.affiliation.instituteInst. Chemiede
tub.publisher.universityorinstitutionTechnische Universität Berlinen
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