Functional Interactions of Lipids for Kir Channels and YidC Insertases via Molecular Dynamics Simulations, SSNMR, and Functional Assays

Inwardly Rectifying Potassium channels (Kir channels) modulate cellular charge gradients as a function of ligand recognition. These ligands include anionic lipids, activated G-proteins, and ATP. The Kir channel KirBac1.1 found in Burkholderia pseudomallei is over 50% homologous to its mammalian counterparts, making it the ideal surrogate protein for dynamic ligand-binding studies. In my work, I have utilized fluorescent based flux assays to investigate how protein-lipid interactions modulate the activity of KirBac1.1. These interactions include channel allostery, interfacial tryptophans, and N-terminal residues. To further expand on these interactions, I present ammonium uptake assays that show KirBac1.1 can utilize NMR friendly 15NH4+ in the selectivity filter. I then utilized ILV as a selective labelling pattern for future SSNMR studies. Finally, I show how DUMB optimization can enhance the study of mammalian Kir channels like hKir2.1, GIRK1, and GIRK2. The YidC family is a class of proteins insertases. YidCs are membrane proteins that are responsible for inserting other membrane proteins into the lipid bilayer. The YidC family is essential to the function and proliferation of Streptococcus mutans. Knowledge of the structure-function relationship of these proteins will greatly inform the pathogenesis of Streptococcus mutans in tooth decay. We developed a fluorescence polarization assay to measure the insertion of the viral coat protein Pf3 by YidC1 and YidC2. We then used this assay to quantify the role of anionic lipids in insertase activity and determine how activity is altered as the pH lowers from 7.5 to 5. Human adenoviruses (HAdV) have been a recent target for obesity research. Early region 4 open reading frame 1 (E4orf1) from HAdV-36 has been the focus on how it alters the metabolism of mice and humans. I have successfully purified the HAdV-36 E4orf1 full construct in a monomer form for use in future studies. I have also created a protocol to purify B1 immunoglobulin binding domain of streptococcal protein G (GB1), a fundamental protein NMR standard. This protocol allows us to purify GB1 with high yield and high purity in a “one pot” purification protocol for future NMR labeling strategies.