Uncoupling of ligand-binding affinity of the bacterial serine chemoreceptor from methylation- and temperature-modulated signaling states.
The Escherichia coli chemoreceptor Tsr mediates tactic responses to serine, repellents, and changes in temperature. We have previously shown that the serine-sensing ability of Tsr-T156C, which has a unique cysteine in place of threonine at residue 156, is specifically inactivated by thiol-modifying reagents and that L-serine protects the receptor from modification. In this study, we demonstrated the correlation between protective effects of various attractants and their potencies to elicit attractant responses. This indirect binding assay was used to monitor the affinity of the receptor for L-serine under various conditions. It has been demonstrated by in vitro assays that the ligand-binding affinities of Tsr and the related chemoreceptor Tar are unaffected by changes in the methylation state of the receptor. Using the serine protection assay, we re-examined this issue both in vitro and in vivo. The methylation levels of Tsr-T156C did not affect its ligand-binding affinity. We also showed both in vitro and in vivo that the ligand-binding affinity was unaffected by temperature. These results suggest that the structure of the periplasmic domain of the receptor is uncoupled from the signaling states of the cytoplasmic domain. This ligand-binding assay system should be applicable to other receptors.
Iwama
T
T
Department of Biotechnology, Division of Utilization of Biological Resource, Faculty of Agriculture, Gifu University, Yanagido, Gifu 501-11, Japan.
Homma
M
M
Kawagishi
I
I
D000577:Amides / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D003553:Cystine / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli; D005033:Ethylmaleimide / Q000008:administration & dosage / Q000494:pharmacology; D066298:In Vitro Techniques; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D008745:Methylation; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013696:Temperature
D000577:Amides; D001426:Bacterial Proteins; D008024:Ligands; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; C057635:Tsr protein, Bacteria; D012694:Serine; D003553:Cystine; D005033:Ethylmaleimide