Using Mutagenesis To Assist In Nmr Assignment Of Chemotaxis Receptor

Chemotaxis is a process that many bacteria use to direct their movement towards or away from chemical stimuli. Some bacteria also use chemotaxis to localize in a host body and thus cause disease. Understanding how the chemotaxis system works will not only provide us with basic knowledge about this complex system, but will also allow us to implement that knowledge to inhibit chemotaxis in pathogenic bacteria and thus maybe prevent disease. Our experiment focused on the chemical chemotaxis receptor protein (TM14), which senses the concentration of the chemicals around the bacteria. To study the structure and interactions of this receptor, our lab uses NMR (nuclear magnetic resonance) spectroscopy. To use the NMR spectrum for interaction and structure analysis, each peak on the spectrum has to be assigned to a specific amino acid on the chemotaxis receptor. To help with the assignment of some difficult-to-assign peaks, we created bacteria that expressed mutant receptors with a single amino acid mutation (the mutated amino acid was the one that we thought to correlate with one of the unassigned peaks; the rest of the receptor was unchanged). We deuterated and labeled certain areas of the mutant receptors with carbon-13 (as the more common carbon-12 isotope is not visible on NMR), and then ran an NMR scan on them. By comparing the NMR spectra of the original wildtype receptor to the mutant receptor, we could verify the assignment of NMR peaks to amino acids. What we saw was that when we mutated a specific amino acid on the chemotaxis receptor, the peak that correlates to that amino acid would disappear. Overall our experiment verified the correct assignments of the peaks we tested.