Synthesis of a Polyhistidine-bearing Amphipol and its Use for Immobilizing Membrane Proteins.
Amphipols (APols) are short amphipathic polymers that stabilize membrane proteins (MPs) in aqueous solutions. In the present study, A8-35, a polyacrylate-based APol, was grafted with hexahistidine tags (His6-tags). The synthesis and characterization of this novel functionalized APol, named HistAPol, are described. Its ability to immobilize MPs on nickel ion-bearing surfaces was tested using two complementary methods, immobilized metal affinity chromatography (IMAC) and surface plasmon resonance (SPR). Compared to a single His6-tag fused at one extremity of a MP, the presence of several His6-tags carried by the APol belt surrounding the transmembrane domain of a MP increases remarkably the affinity of the protein/APol complex for nickel ion-bearing SPR chips, whereas it does not show such a strong effect on an IMAC resin. HistAPol-mediated immobilization, which allows reversibility of the interaction and easy regeneration of the supports and dispenses with any genetic modification of the target protein, provides a novel, promising tool for attaching MPs onto solid supports while stabilizing them.
Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, UMR 7099, CNRS/Université Paris 7, Institut de Biologie Physico-Chimique (FRC 550), 13 rue Pierre et Marie Curie, F-75005 Paris, France.
Fabrice
F
Giusti
CEA, Institut de Biologie et de Technologies de Saclay, Service d'Ingénierie Moléculaire des Protéines, 91191 Gif-sur-Yvette, France.
Pascal
P
Kessler
Department of Chemistry & Nano-Science Center, University of Copenhagen , Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark.
Randi Westh
RW
Hansen
Department of Chemistry & Nano-Science Center, University of Copenhagen , Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark.
Eduardo A
EA
Della Pia
Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, UMR 7099, CNRS/Université Paris 7, Institut de Biologie Physico-Chimique (FRC 550), 13 rue Pierre et Marie Curie, F-75005 Paris, France.
Christel
C
Le Bon
CEA, Institut de Biologie et de Technologies de Saclay, Service d'Ingénierie Moléculaire des Protéines, 91191 Gif-sur-Yvette, France.
Gilles
G
Mourier
Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, UMR 7099, CNRS/Université Paris 7, Institut de Biologie Physico-Chimique (FRC 550), 13 rue Pierre et Marie Curie, F-75005 Paris, France.
Jean-Luc
JL
Popot
Department of Chemistry & Nano-Science Center, University of Copenhagen , Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark.
Karen L
KL
Martinez
Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, UMR 7099, CNRS/Université Paris 7, Institut de Biologie Physico-Chimique (FRC 550), 13 rue Pierre et Marie Curie, F-75005 Paris, France.
Manuela
M
Zoonens
D002413:Cations, Divalent; D002846:Chromatography, Affinity; D006639:Histidine / Q000737:chemistry*; D055767:Immobilized Proteins / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D009532:Nickel / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry*; D011108:Polymers / Q000138:chemical synthesis* / Q000737:chemistry; D011437:Propylamines / Q000138:chemical synthesis* / Q000737:chemistry; D055550:Protein Stability; D012996:Solutions; D020349:Surface Plasmon Resonance; D014867:Water
D002413:Cations, Divalent; C471213:His-His-His-His-His-His; D055767:Immobilized Proteins; D008565:Membrane Proteins; D009842:Oligopeptides; D011108:Polymers; D011437:Propylamines; D012996:Solutions; C499017:amphipol A8-35; D014867:Water; D006639:Histidine; D009532:Nickel