Universität zu Köln

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

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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:	Creators Email ORCID ORCID Put Code Jazi, Atieh Aminian UNSPECIFIED UNSPECIFIED UNSPECIFIED Ploetz, Evelyn UNSPECIFIED orcid.org/0000-0003-0922-875X UNSPECIFIED Arizki, Muhamad UNSPECIFIED UNSPECIFIED UNSPECIFIED Dhandayuthapani, Balasubramaniam UNSPECIFIED UNSPECIFIED UNSPECIFIED Waclawska, Izabela UNSPECIFIED UNSPECIFIED UNSPECIFIED Kramer, Reinhard UNSPECIFIED UNSPECIFIED UNSPECIFIED Ziegler, Christine UNSPECIFIED UNSPECIFIED UNSPECIFIED Cordes, Thorben UNSPECIFIED orcid.org/0000-0002-8598-5499 UNSPECIFIED
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:	Keywords Language INDUCED FLUORESCENCE ENHANCEMENT; PHOTOINDUCED ELECTRON-TRANSFER; ALTERNATING-LASER EXCITATION; CONFORMATIONAL DYNAMICS; CORYNEBACTERIUM-GLUTAMICUM; STRUCTURAL BIOLOGY; BETAINE TRANSPORTER; OPEN COMPLEX; FRET; DYES Multiple languages Biochemistry & Molecular Biology Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/23397