In vivo sulfhydryl modification of the ligand-binding site of Tsr, the Escherichia coli serine chemoreceptor.
The Escherichia coli chemoreceptor Tsr mediates an attractant response to serine. We substituted Cys for Thr-156, one of the residues involved in serine sensing. The mutant receptor Tsr-T156C retained serine- and repellent-sensing abilities. However, it lost serine-sensing ability when it was treated in vivo with sulfhydryl-modifying reagents such as N-ethylmaleimide (NEM). Serine protected Tsr-T156C from these reagents. We showed that [3H]NEM bound to Tsr-T156C and that binding decreased in the presence of serine. By pretreating cells with serine and cold NEM, Tsr-T156C was selectively labeled with radioactive NEM. These results are consistent with the location of Thr-156 in the serine-binding site. Chemical modification of the Tsr ligand-binding site provides a basis for simple purification and should assist further in vivo and in vitro investigations of this chemoreceptor protein.
Iwama
T
T
Department of Molecular Biology, Faculty of Science, Nagoya University, Japan.
Kawagishi
I
I
Gomi
S
S
Homma
M
M
Imae
Y
Y
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000235:genetics
D001426:Bacterial Proteins; D008024:Ligands; D008565:Membrane Proteins; C057635:Tsr protein, Bacteria; D013912:Threonine; D012694:Serine; D003545:Cysteine; D005033:Ethylmaleimide