Examining the mechanism of the methionine E. coli ABC transporter MetNI

Abstract

To maintain chemical homeostasis, cells need to transport substrates against their concentration gradients through the cellular membrane. In all life forms, ATP‐binding‐cassette (ABC) transporters utilize the energy of ATP binding and hydrolysis to drive this crucial process. When these transporters malfunction, diseases such as cystic fibrosis, Stargardt’s disease (vision loss), and drug‐resistant cancers can develop. Currently, the structure of a number of ABC transporters in different states has been solved, providing snapshots of the transporter in action. Functional analyses oftentimes reinforce structural studies, however in some cases the proposed models are in disagreement. Recent studies suggest that individual transporters may have multiple mechanisms for transport and that the mechanisms between transporters vary significantly.MetNI is an ABC transporter that imports methionine in bacterial cells. MetI is the transmembrane domain that passes through the cell’s lipid bilayer, and MetN is the site of ATP binding and hydrolysis. Assisting substrate arrival to MetNI is substrate‐binding protein MetQ. Our research focuses on determining the conditions necessary for the interaction of MetNI and MetQ to occur. Using fluorescence anisotropy and fluorescently labeled MetQ, we measure the change in anisotropy as the concentration of MetNI increases. From the anisotropy data we determine the binding dissociation constant of MetNI‐MetQ in the presence and absence of ATP, ADP, and methionine.