The transmembrane serine receptor Tsr associates with a coupling protein, CheW,
and a histidine kinase, CheA, to form a ternary complex that regulates the activity of
CheA. CheA activity is inhibited by binding of L-serine to Tsr. This work aims to
characterize the ligand-binding properties of Tsr and the inhibitory effect of L-serine on
CheA activity. The periplasmic domain of Tsr (pTsr) was purified and characterized.
Analytical gel filtration and analytical ultracentrifugation indicated that binding of Lserine
promotes dimerization. The binding stoichiometry and dissociation constant for
binding of L-serine to pTsr were determined by fluorescence spectroscopy. As protein
concentration decreased, the dissociation constant increased. A working model was
proposed to account for the interactions between L-serine and pTsr. The activity of
CheA in a ternary complex with full-length Tsr and CheW was analyzed by measuring
the production of [32P]-phospho-CheY. (Phospho-CheY is the product of CheA catalysis.)
The results revealed that binding of L-serine decreased CheA activity without changing
its affinity for ATP. These findings suggest that the allosteric effect of L-serine on CheA activity might occur through V-type inhibition. Optimization of an alternative,
continuous, non-radioactive assay for CheA is underway.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/3930 |
| Date | 16 August 2006 |
| Creators | Fan, Lin |
| Contributors | Reinhart, Gregory D. |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Thesis, text |
| Format | 1681536 bytes, electronic, application/pdf, born digital |
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