Two-dimensional recognition pattern of lipid-anchored Fab' fragments.
A two-dimensional pattern of oriented antibody fragments was formed at the air-water interface and transferred onto a solid support. The Fab'-fragments of a monoclonal antibody against the hapten dinitrophenyl (DNP) were covalently linked via a hydrophilic spacer to phospholipid vesicles. A monomolecular lipid-protein layer at equilibrium with these vesicles was allowed to form at the air-water interface. The monolayer was separated from the vesicle phase and transferred to a Langmuir-Blodgett trough. By cooling and compressing, the previously homogeneous lipid-protein film was driven into a two-dimensional phase separation resulting in protein-rich domains and a second phase consisting mainly of lipid. This film was transferred onto a solid support in a way that preserved the protein-lipid pattern. The specificity as well as the contrast in the binding activity of the two different separated phases were then quantified using microfluorometry. DNP conjugated to fluorescein-labeled bovine serum albumin (BSA) showed virtually no binding to the lipid regions, but gave a ratio of bound DNP-BSA to Fab'-lipid of greater than 50% in the protein-rich domains proving that the Fab'-moiety retained its biological activity. This demonstrates that the technique presented here is well suited to modify different solid surfaces with a pattern of a given biological function. The optional control of lateral packing and orientation of the components in the monolayer makes it a general tool for the reconstitution of supported lipid-protein membranes and might also open new ways for the two-dimensional crystallization of proteins at membranes.
Egger
M
M
Technische Universität München, Physikdepartment E22, Garching, FRG.
Heyn
S P
SP
Gaub
H E
HE
D000911:Antibodies, Monoclonal; D004140:Dinitrophenols; D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D007700:Kinetics; D008051:Lipid Bilayers; D008561:Membrane Fusion; D008968:Molecular Conformation; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D004140:Dinitrophenols; D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D008051:Lipid Bilayers