Jazi, Atieh Aminian and Ploetz, Evelyn and Arizki, Muhamad and Dhandayuthapani, Balasubramaniam and Waclawska, Izabela and Kramer, Reinhard and Ziegler, Christine and Cordes, Thorben (2017) Caging and Photoactivation in Single-Molecule Forster Resonance Energy Transfer Experiments. BIOCHEMISTRY, 56 (14). pp. 2031-2041. ISSN 0006-2960,
Full text not available from this repository. (Request a copy)Abstract
Caged organic fluorophores are established tools for localization-based super-resolution imaging. Their use relies on reversible deactivation of standard organic fluorophores by chemical reduction or commercially available caged dyes with ON switching of the fluorescent signal by ultraviolet (UV) light. Here, we establish caging of cyanine fluorophores and caged rhodamine dyes, i.e., chemical deactivation of fluorescence, for single-molecule Forster resonance energy transfer (smFRET) experiments with freely diffusing molecules. They allow temporal separation and sorting of multiple intramolecular donor acceptor pairs during solution-based smFRET. We use this "caged FRET" methodology for the study of complex biochemical species such as multisubunit proteins or nucleic acids containing more than two fluorescent labels. Proof-of-principle experiments and a characterization of the uncaging process in the confocal volume are presented. These reveal that chemical caging and UV reactivation allow temporal uncoupling of convoluted fluorescence signals from, e.g., multiple spectrally similar donor or acceptor molecules on nucleic acids. We also use caging without UV reactivation to remove unwanted overlabeled species in experiments with the homotrimeric membrane transporter BetP. We finally outline further possible applications of the caged FRET methodology, such as the study of weak biochemical interactions, which are otherwise impossible with diffusion-based smFRET techniques because of the required low concentrations of fluorescently labeled biomolecules.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | INDUCED FLUORESCENCE ENHANCEMENT; PHOTOINDUCED ELECTRON-TRANSFER; ALTERNATING-LASER EXCITATION; CONFORMATIONAL DYNAMICS; CORYNEBACTERIUM-GLUTAMICUM; STRUCTURAL BIOLOGY; BETAINE TRANSPORTER; OPEN COMPLEX; FRET; DYES; |
| Subjects: | 500 Science > 540 Chemistry & allied sciences 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie > Prof. Dr. Christine Ziegler |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 14 Dec 2018 13:10 |
| Last Modified: | 20 Feb 2019 07:17 |
| URI: | https://pred.uni-regensburg.de/id/eprint/1039 |
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