Molecular Mechanisms of Channels and Transporters

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ABSTRACT Knowledge of protein motion is necessary to bridge the gap between structure and function and gain insight into the molecular mechanism of protein machines. My lab studies the function of ion channels and ion-coupled transporters, integral membrane proteins that must undergo structural transitions to regulate the flow of ions (channels) or actively pump substrates (transporters) across biological membrane barriers. A set of distinct but interrelated projects examines the mechanism of secondary active transport, promiscuous multidrug recognition, ion channel selectivity and gating, molecular basis of temperature sensing, allosteric regulation of transporter and channel activity, and how small localized interactions can regulate broader dynamics in membrane proteins. These research questions span time- and length- scales, requiring an array of experimental approaches and a well-developed set of model systems to enable hypothesis driven research. One of our primary tools is NMR spectroscopy, which can simultaneously provide structural, thermodynamic (populations), and kinetic (rates of transitions) data with site-specific resolution. NMR chemical shifts are also highly sensitive to changes in the local environment, providing a direct readout of proton binding, a process that is otherwise difficult to monitor experimentally but central to dissecting proton-coupled transport. Over the past 10 years, my lab has done the painstaking work necessary to develop three completely independent model transporter and channel systems (EmrE, NaK, Shaker-VSD) and establish experimental tools ranging from NMR and molecular biophysics to in vitro and in vivo functional assays. We are now primed to address essential research questions that probe the molecular mechanism of these specific systems but also have broader implications for understanding how protein conformational change is regulated, promiscuity versus specificity in substrate recognition, the complexity of proton-coupled transport, and molecular basis for allosteric regulation of protein function.