Examining the Bacterial Methionine Transporter Utilizing Soluble Lipid Bilayer Systems

ATP-binding cassette (ABC) transporters are ubiquitous across all kingdoms of life. These highly specific pumps translocate substrates across cell membranes through 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 such as cystic fibrosis. 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 such as unstable “substitute” mimetic environments. 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. In preparation for, and as subject to, these kind of analyses, this system has been experimentally proven to be durable in a wide range of experimental conditions, including pH, temperature, and salt concentration

Examining the Bacterial Methionine Transporter Utilizing Soluble Lipid Bilayer Systems

The phospholipid bilayer present in both eukaryotes and prokaryotes regulates the cell’s acquisition of nutrients and excretion of waste. Studies on the complex components of the lipid bilayer have paved the way for learning about the selective permeability of the membrane. It is of great interest to understand how materials are being transported through transmembrane proteins in relation to the electrochemical gradient. Investigation into the mechanistic properties of these transport proteins, particularly ATP-binding cassettes (ABC) transporters, can clarify how substrates are being transported via the binding and hydrolysis of adenosine triphosphate (ATP). The study of ABC transporters is significant in human disease treatment; for example, the alteration of the ATP transport protein domain has been found to lead to multidrug-resistance (Boumendjel, A., 2009) and cystic fibrosis (Mendoza, J., 2007). The overall goal of this project is to compare the activity of the MetNI transporter, a methionine importer, solubilized in detergent to the activity in nanodiscs, a self-contained lipidic environment (Sligar, 2008). First, the membrane-scaffolding protein (MSP) component of nanodiscs MSP3 was successfully bacterially expressed in E. coli cells on a large-scale and then purified by fast protein liquid chromatography (FPLC). Preliminary ATPase assays were conducted on detergent-solubilized MetNI. We calculated that the MetNI transporter isolated in detergent has an average Km of 619 µM and kcat of 4.3 min-1. The comparison of the MetNI ATPase rate in lipidic versus detergent environments will be carried out once MetNI is successfully reconstituted into nanodiscs