The e4-allele isoform of apolipoprotein E (ApoE4) plays a key-role in Alzheimer's disease and cardiovascular pathologies. A large body of evidence supports that conformations of the protein are instrumental in its contribution to function and disease; yet, a comprehensive structural picture of ApoE4 is still missing, largely due to the strong aggregation/oligomerization propensity of the protein, which complicates accessing the monomeric form. Furthermore, ApoE4 contains several segments that are intrinsically disordered and, therefore, may be invisible to conventional structural biology methods. In our lab, we have overcome these complications by harnessing state-of-the-art single-molecule fluorescence spectroscopy and, for the first time, we are able to access the structural ensemble of the monomeric full-length ApoE4 (free in solution, embedded in oligomers, and bound to lipids). Interestingly, under native conditions, ApoE4 adopts multiple conformations - previously unidentified - that coexist in equilibrium. These conformations are highly dynamic and malleable to oligomerization and lipid binding. Our experiments provide a new perspective on the mechanism of lipid binding and set the stage for understanding the interaction with Alzheimer's disease factors.

Small multidrug resistance (SMR) transporters provide an ideal system to study the minimal requirements for active transport across a membrane. EmrE is an E. coli SMR transporter that exports a broad class of polyaromatic cation substrates, thus conferring resistance to drug compounds matching this chemical description. As a secondary active antiporter, EmrE drives the uphill export of each substrate molecule by coupling it to the downhill import of 2 protons across the inner membrane. EmrE is proposed to function via a single-site alternating access model.

Bacterial antibiotic resistance is a growing public health concern. One mechanism of resistance arises through drug export by multidrug resistance transporters. To fully understand the function of these proteins requires multiple structures plus kinetic and thermodynamic data to characterize the transport cycle. NMR offers a unique tool to obtain all of this information. The small size of the small multidrug resistance transporter, EmrE, makes it ideal for such studies. EmrE is a secondary active transporter in E. coli that harnesses the H+ gradient to export a broad range of polyaromatic cations from the cell, thus conferring resistance to drugs of this type. Protein conformational change is required for proper transport, allowing alternating access to either side of the membrane in response to substrate binding. We have solubilized EmrE in isotropic bicelles and have found that two conformations are present under these conditions. These two states are interconverting slowly on the NMR timescale, allowing us to study this transporter in action with atomic resolution.