Katherine A. Henzler-Wildman
Role of protein dynamics in multidrug resistance transport activity
Mechanism of Multidrug Resistance Transport
There is growing recognition that protein motion plays an important role in protein function. Our research investigates this relationship in the small multidrug resistance transporter, EmrE. EmrE is an integral membrane protein that resides in the inner membrane of E. coli. It exports polyaromatic cations, reducing their concentration within the cell. Since many drugs are polyaromatic cations, this contributes to E. coli antibiotic resistance. Active transport requires energy, and EmrE drives drug export via coupled import of protons down their electrochemical gradient across the E. coli inner membrane. Although the details are not known, protein conformational change must occur during the transport cycle, allowing alternating access to either side of the membrane in response to substrate binding. A deeper understanding of transport mechanisms requires atomic-resolution structural knowledge coupled with quantitative dynamic information, including the timescale, amplitude, and directionality of structural change in order to build a “movie” of these protein machines in action.
Nuclear Magnetic Resonance Spectroscopy
Our primary experimental tool is nuclear magnetic resonance spectroscopy, which we use to measure the structure and dynamics of EmrE under conditions where it is actively exchanging between different states in the transport cycle. NMR is uniquely suited to this task since kinetic, thermodynamic, and structural data can be acquired simultaneously and with atomic resolution. By coupling this data with traditional binding and transport assays we can determine the functional importance of the protein states and motions we observe.
EmrE as a Model System
As one of the smallest known active transporters, EmrE provides an ideal model system to study several broader questions that go beyond the molecular details of its transport mechanism.
As a multidrug resistance transporter, EmrE recognizes, binds, and transports a broad class of polyaromatic cation compounds. We are investigating how such a diverse array of substrates are recognized by this small protein, and how all these different substrates trigger the same conformational exchange process necessary for transport.
To investigate the influence of the membrane environment on EmrE transport, we study EmrE solubilized in bicelles as well as in full lipid bilayers. The goal is to understand how different lipid compositions affect the structure and dynamics, and thus the function of an integral membrane protein.