Projects in the Henzler-Wildman Lab investigate how the structure and dynamics of integral membrane proteins contribute to their function. We are particularly interested in the molecular mechanism of transporters and channels. Since large scale conformational changes between different states is a key part of the proposed mechanism for transporters and channels, these are ideal systems for studying the role of protein motion in proper function of integral membrane proteins. We use NMR and other biophysical methods to characterize the timescale, amplitude and direction of structural changes, and combine this data with functional assays to study the mechanism of secondary active transport, multidrug recognition, ion selectivity, channel gating, temperature sensing and allosteric regulation of membrane protein function.
EmrE and Other Small Multidrug Resistance (SMR) Transporters
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Protein conformational change is required for active transport, allowing alternating access to either side of the membrane in order to move a substrate “uphill”. The energy source for secondary transporters is the “downhill” flow of another substrate, often protons. Our research investigates the protein dynamics central to the transport process and the coupling between substrates that drives active transport.
Our recent work suggests that EmrE can act as both a proton-coupled symporter and antiporter of different substrates. This would result in susceptibility (symport) as well as resistance (antiport) since the proton motive force is always inwardly directed in bacteria. This is unprecedented for a transporter and is functionally significant because of the potential to control transport direction by manipulating properties of the small molecule, opening up the possibility of designing drugs that are selectively imported into bacteria by the SMR transporters.
How does EmrE recognize and transport such a diverse set of substrates?
We have shown that properties of the small molecule substrate determine its rate of transport by altering the open-in/open-out exchange rate of the transporter. Do substrate properties control not only the rate, but also the direction of transport? We are using the naturally diverse set of substrates for this promiscuous multidrug transporter to study what chemical features make a “good” substrate that is efficiently transported by EmrE and the correlation between specific chemical properties and the rate and direction of transport. We are also studying how the functional phenotype arises from specific details of substrate-transporter interaction and the consequent effects of that interaction on proton coupling, transporter structure and transporter conformational dynamics. We are also studying whether substrates gain access to the transport pore from the aqueous phase or lipid membrane.
What is the native function of EmrE?
The SMR transporters other than EmrE have not been widely studied. Recent research suggests that SugE and many of the “SMR” family may not even function as drug resistance transporters, but are instead guanidinium exporters. We are performing resistance/susceptibility assays to better characterize the substrate profile of SMR transporters and determine whether any SMR homologs naturally confer susceptibility rather than resistance to any compounds.
How is drug efflux efficiently coupled to proton import in this promiscuous protein?
We are performing detailed measurements of reversal potential to characterize the proton/drug coupling as a function of environmental conditions such as temperature and pH. We are using NMR to quantitatively measure the discrete steps of drug and substrate binding and release, transporter dynamics to build a molecular model of the transporter cycle that we can mathematically model and compare with in vitro assays. We are also using EmrE mutants that disrupt particular steps in the transport cycle to understand how transporter dynamics and protonation state are coupled.
NaK, a bacterial non-selective cation channel
NaK provides an ideal model system for investigating ion selectivity and for comparison with the potassium-selective KcsA channel. Although NaK is a non-selective cation channel, two point-mutations convert it into a potassium-selective channel. We are using NMR to study how atomic-level motions influence ion selectivity and the coupling between channel gates that underlies inactivation in the bacterial NaK channel. Functional studies will test whether the insights obtained from our biophysical studies have the proposed effects on the activity of this bacterial channel.
We are collaborating the with Chanda Lab to study how ion channels sense and respond to temperature. We are testing the heat capacity hypothesis that there is no discrete temperature-sensing domain, and instead temperature-dependent channel gating is triggered by differences in overall solvation of the activated and inactivated states. For this project, it is critical to obtain high resolution information on the water- and lipid-accessible surface and how those surface areas change as a function of channel activation and temperature.