7] - The Design and Development of Tar‐EnvZ Chimeric Receptors

Abstract

Escherichia coli histidine kinases play an essential role in sensing external environmental changes. Since the majority of these are transmembrane proteins, it is believed that their periplasmic domains function as receptor and transduce a signal through the transmembrane domain to their cytoplasmic enzymatic domains. Therefore, it is important to understand how signal transduction modulates the enzymatic activities of histidine kinase across transmembrane. Osmosensor histidine kinase EnvZ and chemoreceptor Tar are well-characterized signal-transducing proteins; a fusion of these two proteins would prove to be an ideal tool not only for characterization of histidine kinase EnvZ, but also, more importantly, as a general approach for studying the molecular mechanism of signal transduction across transmembranes. Tar-EnvZ chimeric protein served as a useful tool to study how the signal modulates enzymatic activities of EnvZ by using a well-defined chemical, aspartate, as a receptor ligand. As more and more genome sequences are being published, the number of identified histidine kinases is rapidly growing. The analysis of these newly identified histidine kinases revealed that the architecture of their cytoplasmic domains is more complex than was perceived based on E. coli histidine kinases. Therefore, chimeric proteins of these histidine kinases with Tar receptor would be helpful to study the mechanism of signal transduction. This chapter describes methods for designing chimeric proteins between a histidine kinase of interest and the Tar receptor and applications of the chimeric protein.