Date of Graduation

Summer 8-31-2022

Document Type

Thesis

Degree Name

Master of Science in Chemistry

College/School

College of Arts and Sciences

Department/Program

Chemistry

First Advisor

Janet Yang

Abstract

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 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 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 data show that the binding affinity between MetQ and MetNI is similar in the presence and absence of L- and D-Met. This suggests that substrate discrimination does not occur at the level of MetQ binding. We next explored how different substrates stimulate MetNI ATPase activity. Surprisingly, our data show that apo-MetQ stimulates MetNI ATPase activity more than either L- or D-Met liganded MetQ. These studies point towards an unexpected, novel model for methionine transport.

Lastly, our studies of MetNI regulation support a model in which the periplasmic MetQ allosterically communicates with the cytoplasmic C2 regulatory domains. Consistent with current models for transinhibition, we find that L-Met liganded MetQ is unable to form a complex with MetNI in the presence of high concentrations of L-Met. In contrast, we find that apo-MetQ remains in complex with MetNI despite the presence of L-Met as an inhibitor. This finding suggests that negative regulation of MetNI may only occur when L-Met is available for import.

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