bm5b01010_si_001.pdf (2.19 MB)
Synthesis of a Polyhistidine-bearing Amphipol and its Use for Immobilizing Membrane Proteins
journal contribution
posted on 2015-12-14, 00:00 authored by Fabrice Giusti, Pascal Kessler, Randi
Westh Hansen, Eduardo A. Della Pia, Christel Le Bon, Gilles Mourier, Jean-Luc Popot, Karen L. Martinez, Manuela ZoonensAmphipols (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.