Functional immobilization of biomembrane fragments on planar waveguides for the investigation of side-directed ligand binding by surface-confined fluorescence.
A method for the functional immobilization of Na,K-ATPase-rich membrane fragments on planar metal oxide waveguides has been developed. A novel optical technique based on the highly sensitive detection of surface-confined fluorescence in the evanescent field of the waveguide allowed us to investigate the interactions of the immobilized protein with cations and ligands. For specific binding studies, a FITC-Na,K-ATPase was used, which had been labelled covalently within the ATP-binding domain of the protein. Fluorophore labels of the surface-bound enzyme can be selectively excited in the evanescent field. A preserved functional activity of the immobilized enzyme was only found when a phospholipid monolayer was preassembled onto the hydrophobic chip surface to form a gentle, biocompatible interface. In situ atomic force microscopy (AFM) was used to examine and optimize the conditions for the lipid and membrane fragment assembly and the quality of the formed layers. The enzyme's functional activity was tested by selective K+ cation binding, interaction with anti-fluorescein antibody 4-4-20, phosphorylation of the protein and binding of inhibitory ligand ouabain. The comparison with corresponding fluorescence intensity changes found in bulk solution provides information about the side-directed surface binding of the Na,K-ATPase membrane fragments. The affinity constants of K+ ions to the Na,K-ATPase was the same for the immobilized and the non-immobilized enzyme, providing evidence for the highly native environment on the surface. The method for the functional immobilization of membrane fragments on waveguide surfaces will be the basis for future applications in pharmaceutical research where advanced methods for exploring the molecular mechanisms of membrane receptor targets and drug screening are required.
Pawlak
M
M
Novartis Pharma AG, Preclinical Safety, DMPK, Basel, Switzerland. michael.pawlak@pharma.novartis.com
Grell
E
E
Schick
E
E
Anselmetti
D
D
Ehrat
M
M
D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry*; D005456:Fluorescent Dyes; D008024:Ligands; D008567:Membranes, Artificial; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013499:Surface Properties
D005456:Fluorescent Dyes; D008024:Ligands; D008567:Membranes, Artificial; D000254:Sodium-Potassium-Exchanging ATPase