We will determine how the lipid bilayer organizes around membrane proteins to regulate vital biological functions, including signal transduction and molecular transport. Many lipids and membrane proteins associate to form platforms called lipid rafts, which are phase-separated from the surrounding membrane. The dynamic structure and functional importance of these intermediate-sized (5-200 nm), non-crystalline assemblies are difficult to characterize. Many pathogenic bacteria organize lipid rafts which can increase virulence and antibiotic resistance. In humans, rafts form to facilitate multiple signaling processes. These processes are, in turn, involved in the pathogenesis of diseases, including Alzheimer?s, Parkinson?s, and heart disease. Atomic- resolution dynamic structural details of these assemblies will broaden our understanding of signaling processes and inform disease etiology. We will confront this problem using solid-state NMR (SSNMR) and functional assays in proteoliposomes and biological membranes. 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. It is activated by anionic lipids (especially cardiolipin) and has a high affinity for saturated lipids, cholesterol, and other lipid microdomain-forming components (including hopanoids from the native organism Burkholderia Pseudomallei). The shared regulatory and structural features between KirBac1.1 and eukaryotic Kir channels have inspired several topics of interest: (a) How does the lipid cardiolipin maximally activate KirBac1.1 and trigger transmembrane allostery? Cardiolipin is an essential functional lipid throughout nature, and understanding membrane allostery will inform not only the mechanism of K+ conductance, but the means of transmembrane communications. (b) What is the locus and mechanism of cholesterol/hopanoid induced channel activation? Understanding this is key to determining both how sterols regulate proteins and how they contribute to bilayer organization. (c) How do functional lipid binding sites nucleate rafts? Cardiolipin, cholesterol, and hopanoids are all associated with modulating protein activity and membrane organization;
our aim i s to understand how they create protein-lipid and lipid-lipid interactions in this process. (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 rafts. How do cellular membranes organize such that Kir channels can be in rafts, 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. Our approach will quantify these interactions on the atomic level to understand how these processes occur and how lipid microdomains assemble within biological membranes. Once we understand how functional lipids regulate proteins and how, in turn, proteins order their lipid environment we can develop novel pharmaceuticals and therapeutics to improve human health.
