Jordan Fenlon, University of Utah
Life Sciences
Tsr, the serine chemoreceptor for E. coli, is a transmembrane protein with a periplasmic sensing domain and cytoplasmic adaptation and kinase control domains. The focus of my research project is Tsr residue A413, located in the cytoplasmic tip of the receptor’s kinase control domain. The project involves characterization of mutant Tsr proteins containing amino acid replacements at residue 413. Based on previous work in the Parkinson lab, this residue is thought to play a key role in Tsr signaling by regulating the dynamic motion of the tip.
To measure the signaling behaviors of the mutant receptors, I used a sensitive in vivo kinase assay, based on fluorescence resonance energy transfer (FRET), (Sourjik et al., 2007). In the presence of serine, the Tsr chemoreceptor deactivates the CheA kinase. By challenging cells with different concentrations of serine and measuring their resultant kinase activity, I can obtain a dose-response curve that reflects the stimulus sensitivity of a mutant receptor.
Using this FRET-based assay, I’ve determined the dose-response behaviors of mutant receptors with amino acid replacements at Tsr-A413. The data were fitted to a Hill equation to determine the serine sensitivity and signaling cooperativity for each receptor. The mutant receptors define five kinase activity classes: locked on, locked off, on-biased, off-biased and balanced (wild-type) output. The size and chemical properties of the mutant amino acid side chains defining each output group should provide new insights into the mechanistic role of residue A413 in Tsr function.