Biochimica et Biophysiea Acta. I Ill4(I 992)45-54 45 © 1992Elsevier SciencePublishe~ B.V. All rights reserved(14)fl5-2736/92/$115.110 Synthetic lipid-anchored receptors based on the binding site of a monoclonal antibody Martin Egger *, Svcn-Pcter Heyn and Hermann E. Gaub Phy~il~ Department E22. Biophysik, TU Miinchen. (;arching (Gernmny) ~ Received 7 May It~ ', !) Key words: Lipid anchoredprotein; Fab fragment;Two dimensional recognition pattern: Membranemimeli~.~ Highly specific ligand receptor interactions generally characterize molecular recognition at cell surfaces and other biological systems. In this study we simulate a membrane receptor by fusing a monoclonal antibody fragment to a phosphollpid. A sullhydryl group in the hinge region of a monocional antibody fragment, .was covalently linked to derivatives of phosphatidylothanolamincs and pbosphotidylserine via three different hydrophilic spacer arms. We investigated and characterized these lipid-anchored Fab-fragments which ,~e have named 'Fab-lipids' in lipo~m~l and monolayer systems. Methods for the monomolecular assembling of such films at the air/water interface aml techniques used for their manipulation are outlined. We describe two possibilities for building a mono~r receptor layer, consisting of two-dimrnsional pattern of oriented Fab-frngments with their artificial hydrophobk anchor embedded in a lipid matrix. In the first method a monomoleculnc film at the air/water interface was allmved to form from a veskulnc suspension and driven into a phase separation, resulting in protein rich domains embedded in a protein depleted phase. This film was transferred onto a solid support in such a way that the established pattern was preserved. Alternatively, a recognition pattern was formed by directly cross-linking the Fab-fragments to preformed planar membranes composed of the reactive spacer-lipids and an inert matrix lipid. Specificity as well as contrast of the binding activity of the receptor layers were quantihed using micrn-fluorimctry. * Present address: Boehrinser Mannheim GmbH, 8132 Tutzing, Germany. Abbreviations: DPPE, 1,2-dlpalmitoyl-sn-glycero-3-phosphatidyleth- anolamine (di- 16:0); DSPE, •,2-distearoyl-sn-glyeero-3-phosphatidyl- elhanolamine (di-18:0): DMPS, 1,2.dimyristoyl-sn-glycero.3.phos- phatidyIserine (di-14:0); DMPC, 1,2-dimyristoyl.sn.glycero-3.phes- phatidylehoilne (di.14:0); PDP-PE, propionyldithiopyridonephos- phalidylethanolamine; MPB-PE, maleimidophenylbutyrylphos- phatidylethanolamine; MB-PE. maleimidobenzoylphosphatidyl- ethanolamine: NBD-PC, |-palmitoyl-2-ll2-[(7-nitrobenz-2-oxa-l,3- diazol-4-yl)aminoklodecanyllphosphatidylcholine; SPDP, N-succi- nimidyl 3-[2-pyridyidithiolpropionate; SMPB, succinimidyl 4-[p- maleimidopheny|]butyrate; MBS, m-maleimidobenzoic acid N-hy- droxysucclnlmide ester: EDC, I-ethyl-3-(3-dimethylaminopropyl) earbodiimide HCI; TxR, Texas red (Sulforhodamine 101): FITC, fluoresceinisothiocyanate; BSA, bovine serum a?~umin; OA, ovalbu- min; DNP, 2,4-dinitrophenol-hapten;TLC. thin-layer chromatog- raphy; Fab, antigen bindingfragment. Correspondence: H.E. Gaub. Physik Department E22, Biophysik. TU Milnchen, 8046 Garching, Germany. Introduction Membrane attachment of eukaryotic proteins via hydrocarbon anchors is attracting considerable interest [!,2]. An increasing number of physiologically impor- tant proteins is known to contain a hydrophobic an- chor, ~ither fatty acids (i.e. myristic, palm;tic acids) or complex glycophospholipi4s (glcosylphosphatidylinosi- tol) [3,4]. The functional role of this modification aside from membrane attachment and dynamics is still un- clear and whether putative recognition processes at cell membranes are involved remains to be revealed [5]. In order to study ~his type of questions we designed a model system from a univalent binding site af a monoclonal antibody to which a variety of lipids were attached (Fab-lipids). The approach presented here has the major advantage that with the help of the monoclonal antibody technology we were supplied with