title: Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro.
abstract: Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.
authors.affiliation: Department of Pharmacology, Center for Mitochondrial Diseases, The Cleveland Center for Membrane and Structural Biology, Case Western Reserve University School of Medicine.
authors.forename: Ryan W
authors.initials: RW
authors.lastname: Clinton
authors.affiliation: Department of Pharmacology, Center for Mitochondrial Diseases, The Cleveland Center for Membrane and Structural Biology, Case Western Reserve University School of Medicine; jason.mears@case.edu.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Mears
mesh_terms: D020558:GTP Phosphohydrolases / Q000737:chemistry; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry; D008869:Microtubule-Associated Proteins / Q000737:chemistry; D024101:Mitochondrial Proteins / Q000737:chemistry; D009571:Nitrilotriacetic Acid; D025941:Protein Interaction Mapping
chemical_list: D008055:Lipids; D008081:Liposomes; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D024101:Mitochondrial Proteins; D020558:GTP Phosphohydrolases; D009571:Nitrilotriacetic Acid