Examining the Bacterial Methionine Transporter Utilizing Soluble Lipid Bilayer Systems

Abstract

ATP‐binding cassette (ABC) transporters are ubiquitous across all kingdoms of life. These highly specific pumps translocate substrates across cell membranes using the energy from ATP binding and hydrolysis. A detailed understanding of ABC transporter mechanism could aid in the treatment of a variety of human disorders in which ABC transporters are defective (eg. Stargardt disease, adrenoleukodystrophy, Tangier disease, and cystic fibrosis). Furthermore, ABC transporters are one of the main culprits behind multidrug resistance, a principle impediment facing current cancer therapeutics. Circumventing these diseases depends largely on our ability to fully understand the mechanism of how these transporters mediate movement across the lipid bilayer.While the structural determinations of ABC transporters have provided critical insights, a detailed molecular understanding of how these proteins work has been precluded by difficulties in the functional study of transporters. This gap in our understanding reflects the experimental challenges to their study. One main issue is preserving a transmembrane (TM) protein's native structure and functionality for study. The native membrane environment is a lipid bilayer, which historically has been difficult to recreate for biochemical study. Processes used to produce membrane vesicles require repeated trial and error attempts to address each protein's unique preferences. A more universal strategy for solubilizing TM proteins is the use of synthetic detergents, however many preceding studies suggest that detergents may alter the structure and functionality of the protein. To address this issue, we have turned to a system called nanodiscs, which is a discoidal lipid bilayer encircled by a protein helical belt. Unlike liposomes, nanodiscs have been shown to be a viable system for fluorescent spectroscopic analysis. This system has been experimentally proven to be durable in a wide range of experimental conditions, including pH, temperature, and salt concentration.As part of the Yang Group at the University of San Francisco, I am probing the mechanism of the E. coli methionine transporter model system. This bacterial transporter, named MetNI, imports the amino acid methionine into the cell using energy from ATP. Importantly, MetNI shares high sequence and structural homology with human transporters known to confer multidrug resistance and disease. Over the last year, we have optimized the protocol to generate monodisperse MetNI nanodiscs in high yield. Now, I am utilizing quantitative methods to understand how the MetNI transporter, embedded in a lipid bilayer, can coordinate the binding of substrate and ATP hydrolysis to drive the transport of L‐methionine across cell membranes.Support or Funding InformationAcknowledgements to the Whitehead Fellowship, Clare Boothe Luce Program, and University of San FranciscoThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.