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


CONCLUSIONS
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.
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India.
Ruchi
R
Agrawal
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India.
Prem
P
Kumar V
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India.
Harini
H
Ramanan
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India; Center for Biosystems Science and Engineering, Indian Institute of Science, Bangalore, India. Electronic address: deepak@mrdg.iisc.ernet.in.
Deepak Kumar
DK
Saini
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015374:Biosensing Techniques; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D011494:Protein Kinases; C075616:kdpD protein, Bacteria
Crosstalk; FRET; Response regulator; Sensor kinase; Two component signalling