ABC transporters are central in many cellular functions including nutrient uptake, signal transduction membrane assembly, and cellular detoxification. Both structural and functional studies have revealed insights into the high-affinity uptake mechanism of the MetNI methionine ABC transporter. Using the energy from ATP binding and hydrolysis, the MetNI-Q system can import L-Met, D-Met, and other methionine derivatives against concentration gradients. Many mechanistic studies of ABC transporter propose a model in which cognate binding proteins sequester substrates in the periplasm and deliver them to the transporter. In contrast, recent in vivo and crystallographic studies of MetNI-Q suggest that some substrates may be able to access the transporter through a solvent accessible tunnel in the cognate binding protein-transporter complex. These studies suggest that the substrate delivery to MetNI transporter may be more flexible than other ABC transport systems.
To test if the MetNI-Q system transport system uses two mechanisms of substrate delivery, we designed a series of experiments to dissect the individual steps in the transport cycle. As the starting point, we have developed a fluorescent anisotropy assay to measure the binding affinity between the MetQ substrate-binding protein and the MetNI transporter in the presence of different methionine derivatives. Our preliminary data show that MetNI binds to apo-MetQ ~10 fold tighter than L-Met liganded MetQ, and additionally may differ in the presence of D-Met. This suggests that different methionine derivatives may utilize discrete transport models.
