CheY can exist in at least two states, (i) the unphosphorylated or apo-CheY, and (ii) the phosphorylated CheY. This phosphorylated state of CheY is due to a phospho-transfer from CheA, a histidine auto-kinase. By developing an ELISA based binding assay I have shown a direct binding between CheY and CheA. Also, I have identified the face of CheY which is involved in this interaction by screening CheY mutants for CheA binding. Mutations which affect CheA binding (shown in red) tend to cluster on a face of CheY, whereas the mutations which do not affect binding, lie away from this face (shown in green).
Another important interaction of CheY occurs with a motor-switch component FliM. To study this interaction in detail, I have combined the powerful tools of genetics with biochemistry. I have isolated dominant suppressor mutants of CheY that compensate for defects in FliM. These suppressor mutants are sequenced, and they are clustered in a region of CheY molecule that overlaps with CheA binding face. In order to test this result biochemically, currently I am measuring the FliM binding ability of suppressor CheY mutants using ELISA binding assay. Another interesting part of this project, under my investigation, is allele specificity shown by some of the cheY suppressor mutants for fliM mutations. Using my ELISA binding assay, I am trying to test if this allele specificity is a direct result of CheY-FliM interaction.
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