Characterization of fluorescein arsenical hairpin (FIAsH) as a probe for single-molecule fluorescence spectroscopy

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dc.contributor.authorFernandes, Dennis D.
dc.contributor.authorBamrah, Jasbir
dc.contributor.authorKailasam, Senthilkumar
dc.contributor.authorGomes, Gregory-Neal W.
dc.contributor.authorLi, Yuchong
dc.contributor.authorWieden, Hans-Joachim
dc.contributor.authorGradinaru, Claudiu C.
dc.date.accessioned2020-02-14T22:43:23Z
dc.date.available2020-02-14T22:43:23Z
dc.date.issued2017
dc.descriptionSherpa Romeo green journal. Open access article. Creative Commons Attribution 4.0 International License (CC BY 4.0) appliesen_US
dc.description.abstractIn recent years, new labelling strategies have been developed that involve the genetic insertion of small amino-acid sequences for specific attachment of small organic fluorophores. Here, we focus on the tetracysteine FCM motif (FLNCCPGCCMEP), which binds to fluorescein arsenical hairpin (FlAsH), and the ybbR motif (TVLDSLEFIASKLA) which binds fluorophores conjugated to Coenzyme A (CoA) via a phosphoryl transfer reaction. We designed a peptide containing both motifs for orthogonal labelling with FlAsH and Alexa647 (AF647). Molecular dynamics simulations showed that both motifs remain solvent-accessible for labelling reactions. Fluorescence spectra, correlation spectroscopy and anisotropy decay were used to characterize labelling and to obtain photophysical parameters of free and peptide-bound FlAsH. The data demonstrates that FlAsH is a viable probe for single-molecule studies. Single-molecule imaging confirmed dual labeling of the peptide with FlAsH and AF647. Multiparameter single-molecule Förster Resonance Energy Transfer (smFRET) measurements were performed on freely diffusing peptides in solution. The smFRET histogram showed different peaks corresponding to different backbone and dye orientations, in agreement with the molecular dynamics simulations. The tandem of fluorophores and the labelling strategy described here are a promising alternative to bulky fusion fluorescent proteins for smFRET and single-molecule tracking studies of membrane proteins.en_US
dc.description.peer-reviewYesen_US
dc.identifier.citationFernandes, D. D., Bamrah, J., Kailasam, S., Gomes, G. W., Li, Y., Wieden, H.-J., & Cradinaru, C. C. (2017). Characterization of fluorescein arsenical hairpin (FIAsH) as a probe for single-molecule fluorescence spectroscopy. Scientific Reports, 7, 13063. https://doi.org/10.1038/s41598-017-13427-8en_US
dc.identifier.urihttps://hdl.handle.net/10133/5680
dc.language.isoen_USen_US
dc.publisherNature Researchen_US
dc.publisher.departmentDepartment of Chemistry and Biochemistryen_US
dc.publisher.facultyArts and Scienceen_US
dc.publisher.institutionUniversity of Torontoen_US
dc.publisher.institutionUniversity of Lethbridgeen_US
dc.publisher.urlhttps://doi.org/10.1038/s41598-017-13427-8en_US
dc.subjectMolecular modellingen_US
dc.subjectSingle-molecule biophysicsen_US
dc.subject.lcshMolecules--Models
dc.subject.lcshFluorescein arsenical hairpin
dc.titleCharacterization of fluorescein arsenical hairpin (FIAsH) as a probe for single-molecule fluorescence spectroscopyen_US
dc.typeArticleen_US
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