Liposome-mediated assembly of receptor signaling complexes.
The reconstitution of membrane-associated protein complexes poses significant experimental challenges. The core signaling complex in the bacterial chemotaxis system is an illustrative example: The soluble cytoplasmic signaling proteins CheW and CheA bind to heterogeneous clusters of transmembrane receptor proteins, resulting in an assembly that exhibits cooperative kinase regulation. An understanding of the basis for the cooperativity inherent in the receptor/CheW/CheA interaction, as well as other membrane phenomena, can benefit from functional studies under defined conditions. To meet this need, a simple method was developed to assemble functional complexes on lipid membranes. The method employs a receptor cytoplasmic domain fragment (CF) with a histidine tag and liposomes that contain a Ni(2+) -chelating lipid. Assemblies of CF, CheW, and CheA form spontaneously in the presence of these liposomes, which exhibit the salient biochemical functions of kinase stimulation, cooperative regulation, and CheR-mediated receptor methylation. Although ligand binding phenomena cannot be studied directly with this approach, other factors that influence kinase stimulation and receptor methylation can be explored systematically, including receptor density and competition among stimulating and inhibiting receptor domains. The template-directed assembly of proteins leads to relatively well-defined samples that are amenable to analysis by a number of methods, including light scattering, electron microscopy, and fluorescence resonance energy transfer. The approach promises to be applicable to many systems involving membrane-associated proteins.
Montefusco
David J
DJ
Department of Chemistry, University of Massachusetts, Amherst, MA, USA.
Asinas
Abdalin E
AE
Weis
Robert M
RM
D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D001671:Biochemistry / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000502:physiology*; D006639:Histidine / Q000737:chemistry; D000071677:Histidine Kinase; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008745:Methylation; D008954:Models, Biological; D008956:Models, Chemical; D009532:Nickel / Q000737:chemistry; D017434:Protein Structure, Tertiary; D015398:Signal Transduction
D001426:Bacterial Proteins; C489103:CheW protein, E coli; D029968:Escherichia coli Proteins; D008055:Lipids; D008081:Liposomes; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D006639:Histidine; D009532:Nickel; D000071677:Histidine Kinase; C000607608:cheA protein, E coli