Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Computational and Molecular Biophysics

Language

English (en)

Date of Award

Spring 3-21-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Katherine A Henzler-Wildman

Abstract

EmrE, an E. coli small multidrug resistance transporter, effluxes a diverse range of toxic polyaromatic cations, thus imparting resistance to drug compounds of this type. Transporters must interconvert between inward- and outward-facing structures during the transport cycle to alternate access of its binding site between the two sides of the membrane. As a secondary active antiporter, EmrE must couple drug binding to this conformational exchange and its energy source, the proton gradient across the inner membrane. The transport cycle involves the import of two protons to drive the export of one substrate molecule. As a multidrug resistance transporter, EmrE has particularly broad specificity and a large variation in affinities for these transported substrates, yet all substrates must trigger the same conformational change between inward- and outward-facing states in order for transport to occur. My research focuses on this coupling between substrate binding and conformational exchange and the functional outcome of this coupling, transport.

As one of the smallest transporters, EmrE serves as an ideal system to study the minimal requirements for multidrug recognition and transport. EmrE transports polyaromatic cations that vary in geometry (i.e. planar vs. tetrahedral), charge (i.e. +1 vs. +2), and overall size. By combining a variety of biophysical techniques, I have investigated the thermodynamics and kinetics of broad substrate recognition and the conformational interconversion that it triggers. In particular, I have focused on two ligand series: i) a series of related tetrahedral ligands with an identical charge of +1, and ii) a series of planar ligands that vary in size and charge. EmrE binds these substrates with affinities that span several orders of magnitude and transports them with varying efficiency. We have directly monitored conformational exchange in substrate-bound EmrE using ZZ-exchange NMR spectroscopy and found that the rate of this key step in transport varies considerably with the identity of the bound ligand. These studies have interesting implications for the energetics of the transport cycle. Substrate binding alters both the ground and transition state energies of EmrE. A common theme has emerged in the literature suggesting that enzymes pre-sample their functional motions in the absence of substrate. However, to be a functional antiporter, EmrE cannot interconvert in the absence of substrate. Thus, the protein alone cannot determine the barrier to exchange between the inward- and outward-facing states, and substrate must play an important role.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7RX992D

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