The localization of the phosphorylation site of BglG, the response regulator of the Escherichia coli bgl sensory system.
BglG, the response regulator of the bgl sensory system, was recently shown to be phosphorylated on a histidine residue. We report here the localization of the phosphorylation site to histidine 208. Localization of the phosphorylated histidine was carried out in two steps. We first engineered BglG derivatives with a specific protease (factor Xa) cleavage site that allowed asymmetric splitting of each prephosphorylated protein to well defined peptides, of which only one was labeled by radioactive phosphate. This allowed the localization of the phosphorylation site to the last 111 residues. Subsequently, we identified the phosphorylated histidine by mutating each of the three histidines located in this region to an arginine and following the ability of the resulting mutants to be in vivo regulated and in vitro phosphorylated by BglF, the bgl system sensor. Histidine 208 was the only histidine which failed both tests. The use of simple techniques to map the phosphorylation site should make this protocol applicable for the localization of phosphorylation sites in other proteins. The bgl system represents a new family of sensory systems. Thus, the mapping reported here is an important step toward the definition of the functional domains involved in the transduction of a signal by the components that constitute systems of this novel family.
Chen
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Department of Molecular Biology, The Hebrew University-Hadassah Medical School, P. O. Box 12272, Jerusalem 91120, Israel.
Engelberg-Kulka
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Amster-Choder
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D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010766:Phosphorylation; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D016601:RNA-Binding Proteins; C064872:antiterminator proteins, Bacteria; D006639:Histidine; D015951:Factor Xa