We will determine how the lipid bilayer organizes around membrane proteins to regulate vital biological functions. In pathogenic bacteria lipid microdomains increase virulence and antibiotic resistance. In humans, microdomains can facilitate multiple signaling processes which can malfunction within disease pathogenesis. Our research program is built around three thematic thrusts: (1) To understand how the lipid environment regulates membrane proteins site- specifically. (2) To determine how membrane proteins, in turn, order their environment. (3) To determine the degree of long-range order and dynamic timescales of these membrane assemblies. Our first target is the KirBac1.1 prokaryotic inward-rectifier K+ (Kir) channel and an array of functional lipids, including synthetic lipids and biological lipid extracts, known to associate with rafts. KirBac1.1 shares many behaviors with eukaryotic Kir channels. The shared regulatory and structural features between KirBac1.1 and eukaryotic Kir channels inspire several topics of interest: (a) How do anionic lipids activate KirBac1.1 and trigger transmembrane allostery? (b) What is the locus and mechanism of cholesterol/hopanoid induced channel activation? (c) How do functional lipid binding sites nucleate microdomains? (d) How does the organization of the annular/nonannular lipid shell act as a secondary regulator of membrane proteins? Kir channels are inactivated by cholesterol but have a high affinity for microdomains. How do cellular membranes organize such that Kir channels can be in microdomains, yet retain activity? (e) What is the long-range order and lifetime of these assemblies? It is still unknown if these assemblies persist on the timescale of signaling processes.
The functional interactions between lipids and membrane proteins, and their organization into lipid microdomains, regulate key biological signaling processes and are involved in the pathogenesis of HIV, Alzheimer?s, Parkinson?s, and Heart diseases. To quantify these interactions structurally using solid-state NMR requires high-quality purification of isotopically enriched bilayer components, including membrane proteins and functional lipids at high quantities. The proposed supplement will enable high-volume purification of lipids and proteins allowing the detailed study of disease-relevant bilayer conditions.
