دورية أكاديمية
Characterization of fluorescein arsenical hairpin (FIAsH) as a probe for single-molecule fluorescence spectroscopy
| العنوان: | Characterization of fluorescein arsenical hairpin (FIAsH) as a probe for single-molecule fluorescence spectroscopy |
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| المؤلفون: | Fernandes, Dennis D., Bamrah, Jasbir, Kailasam, Senthilkumar, Gomes, Gregory-Neal W., Li, Yuchong, Wieden, Hans-Joachim, Gradinaru, Claudiu C. |
| بيانات النشر: | Nature Research Department of Chemistry and Biochemistry Arts and Science University of Toronto University of Lethbridge //doi.org/10.1038/s41598-017-13427-8 |
| سنة النشر: | 2017 |
| المجموعة: | University of Lethbridge Institutional Repository |
| مصطلحات موضوعية: | Molecular modelling, Single-molecule biophysics, Molecules--Models, Fluorescein arsenical hairpin |
| الوصف: | Sherpa Romeo green journal. Open access article. Creative Commons Attribution 4.0 International License (CC BY 4.0) applies ; In 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. ; Yes |
| نوع الوثيقة: | article in journal/newspaper |
| وصف الملف: | application/pdf |
| اللغة: | English |
| العلاقة: | Fernandes, 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/s41598-017-13427-8Test; https://hdl.handle.net/10133/5680Test |
| الإتاحة: | https://hdl.handle.net/10133/5680Test |
| رقم الانضمام: | edsbas.6D86E527 |
| قاعدة البيانات: | BASE |
| الوصف غير متاح. |