FRET reveals multiple interaction states between two component signalling system proteins of M. tuberculosis.

ABSTRACT

BACKGROUND: Two component signalling involves interaction between sensor kinase (SK) and response regulator (RR) proteins which depends on their phosphorylation status. METHODS: In this study we report the development of an in vitro FRET assay for studying interaction between fluorescently tagged SK and RR proteins. RESULTS: Using TCS proteins of Mycobacterium tuberculosis, we demonstrate that phosphorylation status of SK affects the SK-RR interaction, which varies from one TCS to another. The observation was strengthened by recordings from mutant SK and RR proteins. The assay retained the specificity/crosstalk potential of the participating proteins and reflected the inherent phosphotransfer potentials. CONCLUSIONS: SK and RR proteins interact with each other in unphosphorylated state and the phosphorylation affects the interaction between SK and RR, which was reflected as reduction in FRET ratio. GENERAL SIGNIFICANCE: A non-radioactive, in vitro FRET based assay is reported, which can be utilized for studying genome-wide partner screening, identifying crosstalk or specificity in TCSs.