Jazi, Atieh Aminian, Ploetz, Evelyn ORCID: 0000-0003-0922-875X, Arizki, Muhamad, Dhandayuthapani, Balasubramaniam, Waclawska, Izabela, Kramer, Reinhard, Ziegler, Christine and Cordes, Thorben ORCID: 0000-0002-8598-5499 (2017). Caging and Photoactivation in Single-Molecule Forster Resonance Energy Transfer Experiments. Biochemistry, 56 (14). S. 2031 - 2042. WASHINGTON: AMER CHEMICAL SOC. ISSN 0006-2960

Full text not available from this repository.

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: Journal Article
Creators:
CreatorsEmailORCIDORCID Put Code
Jazi, Atieh AminianUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Ploetz, EvelynUNSPECIFIEDorcid.org/0000-0003-0922-875XUNSPECIFIED
Arizki, MuhamadUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Dhandayuthapani, BalasubramaniamUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Waclawska, IzabelaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Kramer, ReinhardUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Ziegler, ChristineUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Cordes, ThorbenUNSPECIFIEDorcid.org/0000-0002-8598-5499UNSPECIFIED
URN: urn:nbn:de:hbz:38-233974
DOI: 10.1021/acs.biochem.6b00916
Journal or Publication Title: Biochemistry
Volume: 56
Number: 14
Page Range: S. 2031 - 2042
Date: 2017
Publisher: AMER CHEMICAL SOC
Place of Publication: WASHINGTON
ISSN: 0006-2960
Language: English
Faculty: Unspecified
Divisions: Unspecified
Subjects: no entry
Uncontrolled Keywords:
KeywordsLanguage
INDUCED FLUORESCENCE ENHANCEMENT; PHOTOINDUCED ELECTRON-TRANSFER; ALTERNATING-LASER EXCITATION; CONFORMATIONAL DYNAMICS; CORYNEBACTERIUM-GLUTAMICUM; STRUCTURAL BIOLOGY; BETAINE TRANSPORTER; OPEN COMPLEX; FRET; DYESMultiple languages
Biochemistry & Molecular BiologyMultiple languages
Refereed: Yes
URI: http://kups.ub.uni-koeln.de/id/eprint/23397

Downloads

Downloads per month over past year

Altmetric

Export

Actions (login required)

View Item View Item