Peptides and synthetic cell membranes

Details Peptides and synthetic cell membranes Peptides and synthetic cell membranes

1997-06-13

1999-10-05

Tsang, Cecilia J.

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

6 to 7 amino acid residues in defined sequence

530328, 530330, 436524, 436525, C07K 700

Patent

active

059626380

DESCRIPTION:

BRIEF SUMMARY
The invention relates to peptides or peptide-analogous compounds of the formula I Leu, Ala, Ser, Gly-Gly and Ser-Ser, consisting of Ala, Ala-Ala, Leu-Leu, Ala-Ala-Ala, Arg-Gly-Asp and Leu-Leu-Leu, Ala, Leu and Pro-Lys, Trt-S-alkyl-CO-NH-alkyl'-CO- or 1,2-dithiocyclopentane-3-(CH.sub.2).sub.4 --CO--, where R can be H only if at least one of the residues A, B, C, D or E is not Ala, radical with 1 to 11 C atoms
Lipids and proteins are major components of biological membranes. Lipid bilayers are regarded as a model of cell membranes. Peptides or proteins can be incorporated into such lipid bilayers so that they extend through them by insertion perpendicular to the surface (J. C. Huschilt et al. BBA 979, 139-141 (1989)). The conformation of such a bilayer is partly determined by the sequence of the peptide. J. D. Leer et al. (Science 240, 1177-1181 (1988)) were able to show that the peptide H-(Leu-Ser-Ser-Leu-Leu-Ser-Leu).sub.3 --CONH.sub.2 (SEQ ID NO:1) with a length which extends through the membrane forms, as amphiphilic alpha helix, an ion channel which arises due to self-organization of these helices in bundles. A peptide consisting of 14 amino acids with this sequence, which is too short to extend through the lipid bilayer, was unable to form this discrete channel. On the other hand, the peptide H-(Leu-Ser-Leu-Leu-Leu-Ser-Leu).sub.3 --CONH.sub.2 (SEQ ID NO:2) forms a proton channel.
Omega-substituted alkanethiols bind and organize themselves on gold surfaces (C. D. Bain et al., Angew. Chemie 101, 522-528 (1989)).
Alkanethiols (octanethiol and hexadecanethiol) form, on gold-coated electrodes, an organized monolayer on which lipid bilayers can be formed with lipids. Adsorption of proteins (cholera toxin) to the monolayer can be analyzed electrochemically (impedance measurement) and optically (surface plasmon resonance). The electrochemical measurement provides information on the quality of the layer and on the amount of ligand molecule inserted. The optical method permits the amount of lipid bound to gold, and the selective binding of the acceptor molecule (cholera toxin) to the membrane, to be quantified (S. Terrettaz et al. Langmuir 9, 1361-1369 (1993)).
The interaction of surface-bound anti-mouse IgG antibodies with mouse IgG as ligand has been measured not on gold but on tantalum/tantalum oxide. The change in impedance with this immunochemical reaction was analyzed in real time in a flow cell. The protein was not anchored via a thiol but bound via an aminoalkylsilane (A. Gebbert et al. Anal. Chem. 64, 997-1003 (1992)).
It was possible to load spherical particles of polymethylmethacrylate with H-(Ala).sub.5 -OMe (SEQ ID NO:3) with the aid of a carbodiimide. After hydrolysis of the esters, it was possible to couple the lipid phosphatidylethanolamine to the carboxyl groups of the peptides. It was possible by adsorption of further phospholipids to form lipid bilayers into which bacteriorhodopsin was incorporated. It was possible to show, by detecting the direction of the proton pumps produced and electron microscopy that the preferred orientation of the bacteriorhodopsin in the lipid layer runs from the inside to the outside (U. Rothe et al. FEBS Lett. 263, 308-312 (1990)).
The invention was based on the object of finding novel compounds with valuable properties, in particular those which can be used to prepare peptide layers, peptide-analogous layers or cell membranes.
It has been found that mercaptoalkylcarboxy-peptides of the formula I are bound to gold surfaces and that they organize themselves to dense layers, especially when lipids (for example dimyristoylphosphatidyloxyethylamine, DMPE) are covalently coupled to the peptidylgold phase.
This produces on the gold surface monomolecular lipid layers which are covalently bonded via the peptide spacer group to the gold. Alternatively, lipid layers can also be applied to the peptide layer by the Langmuir-Boldgett technique. The method is described, for example, by G. Puu, I. Gustavson, P.-A. Ohlsson, G. Olofson and A. Sellstr.o slashed.m in Progress in Memb

REFERENCES:
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Survay, M.. et al. (1993). "Oligoglycines and oligoalanines as tests for modelling mobility of peptides in capillary electrophoresis," Journal of Chromatography, 636, 81-86.
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Lang, H. et al (1994). "A New Class of Thiolipids for the Attachment of Lipid Bilayers on Gold Surfaces," Langmuir, 10, 197-210.
Rothe, U. et al. (1990). "Oriented incorporation of bacteriorhodopsin into the lipid shell of phospholipid-coated polymer particles," FEBS Letters, 263, 308-312.
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