Date of Graduation
Master of Science in Chemistry
College of Arts and Sciences
Janet G. Yang
Membrane transporter proteins play the vital role of moving compounds in and out of the cell and are essential for all living organisms. ATP Binding Cassette (ABC) family transporters function both as importers and exporters in prokaryotes. MetNI is an E. coli Type I ABC transporter responsible for the uptake of methionine into the cytosol from the cell periplasmic space through the cell membrane to maintain intracellular methionine pools. ABC transporters, like other membrane proteins, are most often mechanistically and structurally studied in vitro, solubilized by detergents. However, detergent micelles may affect the conformational changes of membrane proteins relative to those in a native membrane lipid environment, altering transport and ATPase hydrolysis activities. The goal of this research project was to test a promising alternative to liposomal reconstitution of MetNI for preserving native activities. This system, termed “nanodiscs,” is a well-established biochemical tool for membrane proteins consisting of a nanoscale discoidal bilayer of polar lipids encircled by an amphipathic scaffold of apolipoprotein ApoA1 proteins. In this work, the MetNI transporter was purified and successfully reconstituted into two different MSP phospholipid bilayer nanodisc systems (MSP1D1 and MSP1E3D1), confirmed by size exclusion chromatography, native gel electrophoresis, and co-immunoprecipitation. Stabilization of a more native MetNI structure following nanodisc reconstitution was evaluated by the level of basal ATP hydrolysis when compared with purified MetNI solubilized by detergent. Surprisingly, the KM and Vmax of ATP hydrolysis were similar for both forms of MetNI, indicating the presence of basal, futile ATP hydrolysis in both types. Futile ATP hydrolysis was also observed for a mutant MetNI form which lacks methionine substrate inhibition of ATP hydrolysis, MetNI N295A. Additionally, binding of purified substrate binding protein (SBP) to the detergent-solubilized and nanodisc-reconstituted MetNI transporter, was demonstrated by fluorescence anisotropy. Future functional assays performed on completely reconstituted MetNI-SBP complexes in lipid nanodiscs could reveal critical insights into the lipid and structural requirements for native transporter activity
Winslow, Michael T., "Nanodiscs: A novel approach to the study of the Methionine ABC Transporter System" (2023). Master's Theses. 1516.
Amino Acids, Peptides, and Proteins Commons, Biochemistry Commons, Enzymes and Coenzymes Commons, Lipids Commons, Macromolecular Substances Commons, Other Biochemistry, Biophysics, and Structural Biology Commons, Structural Biology Commons