Binding of the Escherichia coli response regulator CheY to its target measured in vivo by fluorescence resonance energy transfer.
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.
Sourjik
Victor
V
Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.
Berg
Howard C
HC
D001426:Bacterial Proteins / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004735:Energy Transfer; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D011993:Recombinant Fusion Proteins; C000607606:cheY protein, E coli; C054562:yellow fluorescent protein, Bacteria; C066821:FliM protein, Bacteria; D049452:Green Fluorescent Proteins