Project Summary All organisms must selectively regulate the uptake and extrusion of molecules from the environment. In addition to identifying and importing crucial ions, nutrients, and transition metals, cells must also rigorously select and export a wide range of substrates, including peptides, polysaccharides, and toxins. The cell employs ATP-binding cassette (ABC) transporters to drive the unidirectional transport of substrates against their concentration gradients. This proposal seeks to understand how prokaryotic ABC importers uptake nutrients that are crucial for survival, and these findings could provide new targets for treatment against bacterial pathogens. Towards this end, we will employ biochemical and biophysical methods to dissect how the E. coli MetNI transporter, an established model system, transports methionine from the periplasm to the cytoplasm. While the most widely- accepted model for import is based on structural studies, emerging functional studies suggest a substantially different model for substrate translocation. These disparate models appear to conflict; however, we hypothesize that the MetNI transporter can adopt multiple modes of transport in response to substrate features and availability. We will use the tools of mechanistic enzymology to resolve these longstanding issues and to propose new models that may merge previous findings or uncover entirely new mechanisms of action. In Aim 1, we will test our hypothesis by measuring the kinetic and thermodynamic parameters of individual steps in the transport cycle. Using different substrates (L-Met, D-Met, and larger methionine derivatives) and binding-impaired mutants, we will dissect three steps: (a) binding of the MetQ cognate periplasmic protein to the MetNI transporter, (b) ATP binding and hydrolysis, and (c) transport. Our findings could reveal that the MetNI-Q mechanism is inherently versatile and could preferentially select different translocation pathways depending on the substrate. For Aim 2, we have generated heterodimeric MetNI “chimeras” to decipher if the two identical ATP sites work together to enable efficient and specific transport. Specifically, we will determine if one or two ATP molecules are hydrolyzed per transport cycle, and if transport efficiency is affected by impairment of one ATP site. Overall, our findings will yield critical insights into how ABC transporters select for different substrates, and how ATP usage drives substrate translocation across membranes.