Nanostructures for Solubilizing Serpentine Receptors

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DESCRIPTION (provided by applicant): Membrane proteins represent a large proportion of all cellular proteins, play critical roles in cellular processes and are involved in many aspects of human disease. A difficulty in membrane protein research in general is that this class of proteins is difficult to express in recombinant form, purify, and characterize structurally through x-ray crystallography or other techniques. The problem arises from the necessity of maintaining mixed polar and hydrophobic environments to maintain the structural and functional integrity of the protein. We have discovered a nanoscopic phospholipid assembly for solubilizing membrane proteins that provides beneficial aspects of both liposomes and detergent micelles. The technology is based on bilayer assemblies of phospholipid and a membrane scaffold protein (MSP) which self-assemble to form nanoscopic phospholipid bilayer disks with the MSP stabilizing the particle at the perimeter of the bilayer domain. Insertion of membrane proteins into the bilayer results in monomeric solubilized species which can be studied in solution or on surfaces. Of particular interest is the solubilization and stabilization of G-protein coupled receptors (GPCR's), or serpentine receptors, which are components of signal transduction. GPCR's are an important and diverse class of receptors that bind several classes of bioactive ligands of pharmacological interest. The experimental strategy for development of the nanobilayer assembly includes molecular engineering aimed at minimizing the structure of the MSP and increasing the stability and monodispersity of the nanobilayer entity by altering the structure of the parent molecule. The MSP is a modified form of human apolipoprotein A-I, a component of circulatory high-density lipoproteins that play a role in cholesterol transport and atherosclerosis. Our discovery also has implications for understanding the structure of lipoproteins as well. The MSP-bilayer structure will also be engineered to allow manipulation of incorporated membrane proteins on solid supports for study by the surface-sensitive techniques of scanning probe microscopy and surface plasmon resonance. The materials and techniques developed will be potentially useful in areas of biotechnology and biological research for structure/function correlation, structure determination, bioseparation, and drug discovery.