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Nonfluorescent quenchers to correlate single-molecule conformational and compositional dynamics.

ABSTRACT: Single-molecule Förster resonance energy transfer (smFRET) is a powerful method for studying the conformational dynamics of a biomolecule in real-time. However, studying how interacting ligands correlate with and regulate the conformational dynamics of the biomolecule is extremely challenging because of the availability of a limited number of fluorescent dyes with both high quantum yield and minimal spectral overlap. Here we report the use of a nonfluorescent quencher (Black Hole Quencher, BHQ) as an acceptor for smFRET. Using a Cy3/BHQ pair, we can accurately follow conformational changes of the ribosome during elongation in real time. We demonstrate the application of single-color FRET to correlate the conformational dynamics of the ribosome with the compositional dynamics of tRNA. We use the normal Cy5 FRET acceptor to observe arrival of a fluorescently labeled tRNA with a concomitant transition of the ribosome from the locked to the unlocked conformation. Our results illustrate the potential of nonfluorescent quenchers in single-molecule correlation studies.

SUBMITTER: Chen J

PROVIDER: S-EPMC4369913 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Nonfluorescent quenchers to correlate single-molecule conformational and compositional dynamics.

Chen Jin J Tsai Albert A Petrov Alexey A Puglisi Joseph D JD

Journal of the American Chemical Society 20120323 13

Single-molecule Förster resonance energy transfer (smFRET) is a powerful method for studying the conformational dynamics of a biomolecule in real-time. However, studying how interacting ligands correlate with and regulate the conformational dynamics of the biomolecule is extremely challenging because of the availability of a limited number of fluorescent dyes with both high quantum yield and minimal spectral overlap. Here we report the use of a nonfluorescent quencher (Black Hole Quencher, BHQ) ...[more]

PMID: 22428667

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Project description:Riboswitches represent a family of highly structured regulatory elements found primarily in the leader sequences of bacterial mRNAs. They function as molecular switches capable of altering gene expression; commonly, this occurs via a conformational change in a regulatory element of a riboswitch that results from ligand binding in the aptamer domain. Numerous studies have investigated the ligand binding process, but little is known about the structural changes in the regulatory element. A mechanistic description of both processes is essential for deeply understanding how riboswitches modulate gene expression. This task is greatly facilitated by studying all aspects of riboswitch structure/dynamics/function in the same model system. To this end, single-molecule fluorescence resonance energy transfer (smFRET) techniques have been used to directly observe the conformational dynamics of a hydroxocobalamin (HyCbl) binding riboswitch (env8HyCbl) with a known crystallographic structure.1 The single-molecule RNA construct studied in this work is unique in that it contains all of the structural elements both necessary and sufficient for regulation of gene expression in a biological context. The results of this investigation reveal that the undocking rate constant associated with the disruption of a long-range kissing-loop (KL) interaction is substantially decreased when the ligand is bound to the RNA, resulting in a preferential stabilization of the docked conformation. Notably, the formation of this tertiary KL interaction directly sequesters the Shine-Dalgarno sequence (i.e., the ribosome binding site) via base-pairing, thus preventing translation initiation. These results reveal that the conformational dynamics of this regulatory switch are quantitatively described by a four-state kinetic model, whereby ligand binding promotes formation of the KL interaction. The results of complementary cell-based gene expression experiments conducted in Escherichia coli are highly correlated with the smFRET results, suggesting that KL formation is directly responsible for regulating gene expression.

Project description:Dark quenchers are chromophores that primarily relax from the excited state to the ground state nonradiatively (i.e., are dark). As a result, they can serve as acceptors for Förster resonance energy transfer experiments without contributing significantly to background in the donor-emission channel, even at high concentrations. Although the advantages of dark quenchers have been exploited for ensemble bioassays, no systematic single-molecule study of dark quenchers has been performed, and little is known about their photophysical properties. Here, we present the first systematic single-molecule study of dark quenchers in conjunction with fluorophores and demonstrate the use of dark quenchers for monitoring multiple interactions and distances in multichromophore systems. Specifically, using double-stranded DNA standards labeled with two fluorophores and a dark quencher (either QSY7 or QSY21), we show that the proximity of a fluorophore and dark quencher can be monitored using the stoichiometry ratio available from alternating laser excitation spectroscopy experiments, either for single molecules diffusing in solution (using a confocal fluorescence) or immobilized on surfaces (using total-internal-reflection fluorescence). The latter experiments allowed characterization of the dark-quencher photophysical properties at the single-molecule level. We also use dark-quenchers to study the affinity and kinetics of binding of DNA Polymerase I (Klenow fragment) to DNA. The measured properties are in excellent agreement with the results of ensemble assays, validating the use of dark quenchers. Because dark-quencher-labeled biomolecules can be used in total-internal-reflection fluorescence experiments at concentrations of 1 ?M or more without introducing a significant background, the use of dark quenchers should permit single-molecule Förster resonance energy transfer measurements for the large number of biomolecules that participate in interactions of moderate-to-low affinity.

Dataset Information

Nonfluorescent quenchers to correlate single-molecule conformational and compositional dynamics.

Publications

Nonfluorescent quenchers to correlate single-molecule conformational and compositional dynamics.

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