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Binding of the Escherichia coli response regulator CheY to its target measured in vivo by fluorescence resonance energy transfer.

ABSTRACT: In Escherichia coli chemotaxis, signaling depends on modulation of the level of phosphorylation of CheY, a small protein that couples receptors and flagellar motors. Working in vivo, we used fluorescence resonance energy transfer (FRET) to measure the interaction of CheY approximately P with its target, FliM. Binding of CheY approximately P to FliM was found to be much less cooperative than motor switching; however, under the conditions of our experiment, most of the FliM appeared to be in the cytoplasm. We studied signal processing times in the chemotaxis pathway by measuring the changes in CheY approximately P binding to FliM on flash release of caged chemoeffectors. Following sudden addition of attractant, the amount of CheY approximately P bound to FliM decayed exponentially with a rate constant of about 2 s(-1). Following sudden addition of repellent, FliM occupancy increased with a rate constant of about 20 s(-1). Using these data, we were able to construct a simple model for the chemotactic pathway and to estimate values of rate constants for several key reactions.

SUBMITTER: Sourjik V

PROVIDER: S-EPMC130518 | biostudies-literature | 2002 Oct

REPOSITORIES: biostudies-literature

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Binding of the Escherichia coli response regulator CheY to its target measured in vivo by fluorescence resonance energy transfer.

Sourjik Victor V Berg Howard C HC

Proceedings of the National Academy of Sciences of the United States of America 20020913 20

In Escherichia coli chemotaxis, signaling depends on modulation of the level of phosphorylation of CheY, a small protein that couples receptors and flagellar motors. Working in vivo, we used fluorescence resonance energy transfer (FRET) to measure the interaction of CheY approximately P with its target, FliM. Binding of CheY approximately P to FliM was found to be much less cooperative than motor switching; however, under the conditions of our experiment, most of the FliM appeared to be in the c ...[more]

PMID: 12232047

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Project description:Conjugated polydiacetylene (PDA) possessing stimuli-responsive properties has been intensively investigated for developing efficient sensors. We report here fluorescence resonance energy transfer (FRET) in liposomes synthesized using different molar ratios of dansyl-tagged diacetylene and diacetylene-carboxylic acid monomers. Photopolymerization of diacetylene resulted in cross-linked PDA liposomes. We used steady-state electronic absorption, emission, and fluorescence anisotropy (FA) analysis to characterize the thermal-induced FRET between dansyl fluorophores (donor) and PDA (acceptor). We found that the monomer ratio of acceptor to donor ( R ad) and length of linkers (functional part that connects dansyl fluorophores to the diacetylene group in the monomer) strongly affected FRET. For R ad = 10 000, the acceptor emission intensity was amplified by more than 18 times when the liposome solution was heated from 298 to 338 K. A decrease in R ad resulted in diminished acceptor emission amplification. This was primarily attributed to lower FRET efficiency between donors and acceptors and a higher background signal. We also found that the FRET amplification of PDA emissions after heating the solution was much higher when dansyl was linked to diacetylene through longer and flexible linkers than through shorter linkers. We attributed this to insertion of dansyl in the bilayer of the liposomes, which led to an increased dansyl quantum yield and a higher interaction of multiple acceptors with limited available donors. This was not the case for shorter and more rigid linkers where PDA amplification was much smaller. The present studies aim at enhancing our understanding of FRET between fluorophores and PDA-based conjugated liposomes. Furthermore, receptor tagged onto PDA liposomes can interact with ligands present on proteins, enzymes, and cells, which will produce emission sensing signal. Therefore, using the present approach, there exist opportunities for designing FRET-based highly sensitive and selective chemical and biochemical sensors.

Dataset Information

Binding of the Escherichia coli response regulator CheY to its target measured in vivo by fluorescence resonance energy transfer.

Publications

Binding of the Escherichia coli response regulator CheY to its target measured in vivo by fluorescence resonance energy transfer.

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