Major
Chemistry
Research Abstract
Intracellular chemical homeostasis is vital for cell survival. A key component for the maintenance of this homeostasis is ABC (ATP-binding cassette) transporters. These highly-conserved transporters utilize the energy from ATP binding and hydrolysis to move a wide array of substrates against their chemical gradient. Mutations can lead to a variety of diseases including cystic fibrosis and drug-resistant tumors. In order to solve these issues, these transporters must be well-studied. This project focuses on the mechanism of the methionine importer MetNI in E. coli, a reliable model for many transporters. Using fluorescence anisotropy, the apo-form of the substrate-binding protein (SBP) was found to have a higher affinity for the ATP-bound transporter than substrate-bound SBP. These results match with that of published data that utilized thermophoresis titration, validating the use of fluorescence anisotropy in assessing binding affinity. Going forward, fluorescence anisotropy will be utilized to assess the binding affinity of methionine to the inhibitory domains of the transporter as well as how complex formation changes if the transporter is only able to bind one ATP molecule.
Faculty Mentor/Advisor
Janet Yang
Included in
Investigating the mechanism of the E.coli ATP-binding cassette transporter MetNI
Intracellular chemical homeostasis is vital for cell survival. A key component for the maintenance of this homeostasis is ABC (ATP-binding cassette) transporters. These highly-conserved transporters utilize the energy from ATP binding and hydrolysis to move a wide array of substrates against their chemical gradient. Mutations can lead to a variety of diseases including cystic fibrosis and drug-resistant tumors. In order to solve these issues, these transporters must be well-studied. This project focuses on the mechanism of the methionine importer MetNI in E. coli, a reliable model for many transporters. Using fluorescence anisotropy, the apo-form of the substrate-binding protein (SBP) was found to have a higher affinity for the ATP-bound transporter than substrate-bound SBP. These results match with that of published data that utilized thermophoresis titration, validating the use of fluorescence anisotropy in assessing binding affinity. Going forward, fluorescence anisotropy will be utilized to assess the binding affinity of methionine to the inhibitory domains of the transporter as well as how complex formation changes if the transporter is only able to bind one ATP molecule.
