title: Single particle tracking reveals corralling of a transmembrane protein in a double-cushioned lipid bilayer assembly.
abstract: A predominate question associated with supported bilayer assemblies containing proteins is whether or not the proteins remain active after incorporation. The major cause for concern is that strong interactions with solid supports can render the protein inactive. To address this question, a large transmembrane protein, the serotonin receptor, 5HT(3A), has been incorporated into several supported membrane bilayer assemblies of increasing complexity. The 5HT(3A) receptor has large extracellular domains on both sides of the membrane, which could cause strong interactions. The bilayer assemblies include a simple POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) supported planar bilayer, a “single-cushion” POPC bilayer with a PEG (poly(ethylene glycol)) layer between membrane and support, and a “double-cushion” POPC bilayer with both a PEG layer and a layer of BSA (bovine serum albumin). Single-cushion systems are designed to lift the bilayer from the surface, and double-cushion systems are designed to both lift the membrane and passivate the solid support. As in previously reported work, protein mobilities measured by ensemble fluorescence recovery after photobleaching (FRAP) are quite low, especially in the double-cushion system. But single-particle tracking of fluorescent 5HT(3A) molecules shows that individual proteins in the double-cushion system have quite high local mobilities but are spatially confined within small corralling domains (<r(C)2> 450 nm). Comparisons with the simple POPC membrane and the single-cushion POPC−PEG membrane reveal that BSA both serves to minimize interactions with the solid support and creates the corrals that reduce the long-range (ensemble averaged) mobility of large transmembrane proteins. These results suggest that in double-cushion assemblies proteins with large extra-membrane domains may remain active and unperturbed despite low bulk diffusion constants.
authors.affiliation: Department of Chemistry, Washington State University, PO Box 644630, Pullman, Washington 99164-4630, United States.
authors.forename: Kumud R
authors.initials: KR
authors.lastname: Poudel
authors.forename: David J
authors.initials: DJ
authors.lastname: Keller
authors.forename: James A
authors.initials: JA
authors.lastname: Brozik
mesh_terms: D000818:Animals; D002417:Cattle; D036681:Fluorescence Recovery After Photobleaching; D055767:Immobilized Proteins / Q000737:chemistry* / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry* / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000737:chemistry
chemical_list: D055767:Immobilized Proteins; D008051:Lipid Bilayers; D044406:Receptors, Serotonin, 5-HT3; D012710:Serum Albumin, Bovine; D011092:Polyethylene Glycols