Oriented attachment and membrane reconstitution of His-tagged cytochrome c oxidase to a gold electrode: in situ monitoring by surface-enhanced infrared absorption spectroscopy.
A novel concept is introduced for the oriented incorporation of membrane proteins into solid supported lipid bilayers. Recombinant cytochrome c oxidase solubilized in detergent was immobilized on a chemically modified gold surface via the affinity of its histidine-tag to a nickel-chelating nitrilo-triacetic acid (NTA) surface. The oriented protein monolayer was reconstituted into the lipid environment by detergent substitution. The individual steps of the surface modification, including (1) chemical modification of the gold support, (2) adsorption of the protein, and (3) reconstitution of the lipid bilayer, were followed in situ by means of surface-enhanced infrared absorption spectroscopy (SEIRAS) and accompanied by normal-mode analysis. The high surface sensitivity of SEIRAS allows for the identification of each chemical reaction process within the monolayer at the molecular level. Finally, full functionality of the surface-tethered cytochrome c oxidase was demonstrated by cyclic voltammetry after binding of the natural electron donor cytochrome c.
Ataka
Kenichi
K
Forschungszentrum Jülich, IBI-2: Structural Biology, Jülich, Germany.
Giess
Frank
F
Knoll
Wolfgang
W
Naumann
Renate
R
Haber-Pohlmeier
Sabina
S
Richter
Björn
B
Heberle
Joachim
J
D004563:Electrochemistry; D003576:Electron Transport Complex IV / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry; D008958:Models, Molecular; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013499:Surface Properties
D004800:Enzymes, Immobilized; D008051:Lipid Bilayers; D011994:Recombinant Proteins; D006639:Histidine; D006046:Gold; D003576:Electron Transport Complex IV; D009571:Nitrilotriacetic Acid