Abstract
BackgroundTwo component signalling involves interaction between sensor kinase (SK) and response regulator (RR) proteins which depends on their phosphorylation status. MethodsIn this study we report the development of an in vitro FRET assay for studying interaction between fluorescently tagged SK and RR proteins. ResultsUsing 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. ConclusionsSK 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 significanceA 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.
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