| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | April 15, 2003 |
| PATENT TITLE |
Stable macroscopic membranes formed by self-assembly of amphiphilic peptides and uses therefor |
| PATENT ABSTRACT |
Described herein is the self-assembly of amphiphilic peptides, i.e., peptides with alternating hydrophobic and hydrophilic residues, into macroscopic membranes. The membrane-forming peptides are greater than 12 amino acids in length, and preferably at least 16 amino acids, are complementary and are structurally compatible. Specifically, two peptides, (AEAEAKAK).sub.2 (ARARADAD).sub.2, were shown to self-assemble into macroscopic membranes. Conditions under which the peptides self-assemble into macroscopic membranes and methods for producing the membranes are also described. The macroscopic membranes have several interesting properties: they are stable in aqueous solution, serum, and ethanol, are highly resistant to heat, alkaline and acidic pH, chemical denaturants, and proteolytic digestion, and are non-cytotoxic. The membranes are potentially useful in biomaterial applications such as slow-diffusion drug delivery systems, artificial skin, and separation matrices, and as experimental models for Alzheimer's disease and scrapie infection. The sequence of the peptide, EAK16, was derived from a putative Z-DNA binding protein from yeast, called zuotin. The cloning and characterization of the ZUO1 gene are also described. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | July 22, 1997 |
| PATENT REFERENCES CITED |
Zhang et al., "Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae", The EMBO J. 11(10):3787-3796 (1992). Osterman, D.G. and Kaiser, E.T., "Design and Characterization of Peptides with Amphiphilic .beta.-Strand Structures", J. Cell. Biochem. 29:57-72 (1985). Brack, A. and Orgel, L.E., ".beta. structures of alternating polypeptides and their possible prebiotic significance", Nature 256:383-387 (1975). Brack, A. and Caille, A., "Synthesis and .beta.-Conformation of Copolypeptides with Alternating Hydrophilic and Hydrophobic Residues", Int. J. Peptide Protein Res. 11:128-139 (1978). Brack, A. and Barbier, B., "Early Peptidic Enzymes", Adv. Spac. Res. 9(6):(6)83-(6)87 (1989). Marqusee S. and Baldwin, R.L., "Helix stabilization by Glu.sup.-. . . Lys.sup.+ salt bridges in short peptides of de novo design", Proc. Natl. Acad. Sci. USA 84:8898-8902 (1987). Marqusee, S., et al., "Unusually stable helix formation in short alanine-based peptides", Proc. Natl. Acad. Sci. USA 86:5286-5290 (1989). Padmanabhan, S., et al., "Relative helix-forming tendencies of nonpolar amino acids", Nature 344:268-270 (1991). Seipke, G., et al., "Synthesis and Properties of Alternating Poly(Lys-Phe) and Comparison with the Random Copolymer Poly(Lys.sup.51, Phe.sup.49)", Biopolymers 13:1621-1633 (1974). St. Pierre, S., et al., "Conformational Studies of Sequential Polypeptides Containing Lysine and Tyrosine", Biopolymers 17:1837-1848 (1978). Peggion, E., et al., "Conformational Studies on Polypeptides. The Effect of Sodium Perchlorate on the Conformation of Poly-L-lysine and of Random Copolymers of L-Lysine and L-Phenylalanine in Aqueous Solution", Biopolymers 11:633-643 (1972). Trudelle, Y., "Conformational study of the sequential (Tyr-Glu).sub..eta. copolymer in aqueous solution", Polymer 16:9-15 (1975). Rippon, W.B., et al., "Spectroscopic Characterization of Poly(Glu-Ala)", J. Mol. Biol. 75:369-375 (1973). Gay, N.J., et al., "A leucine-rich repeat peptide derived from the Drosophila Toll receptor forms extended filaments with a .beta.-sheet structure", FEBS 291(1):87-91 (1991). Hilbich, C., et al., "Aggregation and Secondary Structure of Synthetic Amyloid .beta.A4 Peptides of Alzheimer's Disease", J. Mol. Biol. 218:149-163 (1991). Halverson, K., "Molecular Determinants of Amyloid Deposition in Alzheimer's Disease: Conformational Studies of Synthetic .beta.-Protein Fragments", Biochemistry 29:2639-2644 (1990). Lizardi, P.M., "Genetic Polymorphism of Silk Fibroin Studied by Two-Dimensional Translation Pause Fingerprints", Cell 18:581-589 (1979). Thomas, E.L., "Gigamolecules in Flatland", Science 259:43-45 (1993). Smith, G.G. and Peck, G.E., "Continuous-Flow System for Determination of Diffusion Coefficients: Use of a Natural Membrane", J. of Pharmaceutical Sciences 65(5):727-732 (1976). Hynes, R.O., "Integrins: Versatility, Modulation, and Signaling in Cell Adhesion", Cell 69:11-25 (1992). Yamada, K.M., "Adhesive Recognition Sequences", J. of Biological Chemistry 266(20):12809-12812 (1991). WPI Accession No. 92-313679/38 (JP 4-221395) (1992). WPI Accession No. 92-313678/38 (JP-4-221394) (1992). |
| PATENT GOVERNMENT INTERESTS |
GOVERNMENT FUNDING This work-was supported by the National Institutes of Health, National Science Foundation, National Aeronautics and Space Administration, and Office of Naval Research. The U.S. Government has certain rights in this invention |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A macroscopic membrane which is formed by self-assembly of amphiphilic peptides in an aqueous solution containing monovalent metal cations, wherein the peptides have alternating hydrophobic and hydrophilic amino acids and are complementary and structurally compatible. 2. The membrane of claim 1 wherein the peptides are homogeneous. 3. The membrane of claim 1 which is non-cytotoxic to mammalian cells. 4. A method for forming a macroscopic membrane comprising forming an aqueous mixture of peptides have alternating nonpolar and hydrophilic amino acids that are complementary and structurally compatible, and monovalent metal cations under conditions suitable for self-assembly of the peptides into the macroscopic membrane and allowing the membrane to be formed. 5. The method of claim 4 wherein the peptides are homogenous. 6. The method of claim 4 wherein the peptides are chemically synthesized. 7. The method of claim 4 wherein the monovalent metal cations are selected from Li.sup.+, Na.sup.+, and K.sup.+. 8. The method of claim 4 wherein the peptides are added to an aqueous solution containing the monovalent metal cations. 9. The method of claim 8 wherein the aqueous solution is phosphate-buffered saline. 10. The method of claim 4 wherein the suitable conditions comprise the absence of an inhibitor of the self-assembly of the peptides into the macroscopic membrane. 11. The method of claim 10 wherein the inhibitor is a divalent metal cation. 12. The method of claim 10 wherein the inhibitor is sodium dodecyl sulfate. 13. The method of claim 4 wherein the suitable conditions comprise a pH of less than 12. 14. The membrane of claim 1 wherein the hydrophilic amino acids are acidic and basic amino acids. 15. The membrane of claim 14 wherein the acidic amino acids are independently selected from the group consisting of aspartic acid and glutamic acid. 16. The membrane of claim 15 wherein the basic amino acids are independently selected from the group consisting of arginine, lysine, histidine and ornithine. 17. The membrane of claim 16 wherein the hydrophobic amino acids are selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, serine, threonine and glycine. 18. The membrane of claim 17 wherein the hydrophobic amino acids are alanine. 19. The membrane of claim 1 wherein the peptides are soluble in aqueous solution in the absence of monovalent metal cations. 20. The membrane of claim 1 which is composed of .beta.-sheets. 21. The membrane of claim 1 wherein the difference in interpeptide distance of the complementary peptides upon self-assembly is less than 3 .ANG.. 22. The membrane of claim 21 wherein the interpeptide distance is constant. 23. The membrane of claim 1 wherein the peptides are chemically synthesized. 24. The membrane of claim 1 wherein the monovalent metal cations are selected from the group consisting of Li.sup.+, Na.sup.+, K.sup.+ and Cs.sup.+. 25. The membrane of claim 24 wherein the monovalent metal cation is Na.sup.+. 26. The membrane of claim 25 wherein the monovalent metal cation is added at a concentration of at least 5 mM. 27. The membrane of claim 1 wherein the peptides are present in the aqueous solution at a concentration of at least 1 mg/ml. 28. The membrane of claim 27 wherein the peptides are present in the aqueous solution at a concentration of at least about 10 mg/ml. 29. The membrane of claim 27 wherein the peptide is added to an aqueous solution containing monovalent metal cations. 30. The membrane of claim 1 wherein the membrane is stable in water at a temperature below the boiling point of the water. 31. The membrane of claim 30 wherein the CD spectra of the membrane is unaffected in water at a temperature below the boiling point of water. 32. The membrane of claim 1 wherein the membrane is unaffected at a pH from 1.5 to about 11. 33. The membrane of claim 32 wherein the pH profile of the membrane shows less than 10% decrease in ellipticity of the CD spectra by the treatment. 34. The membrane of claim 33 wherein the membrane is stable in aqueous solution containing less than about 10% sodium dodecyl sulfate, less than 7 M guanidine hydrochloride, or less than 8 M urea. 35. The membrane of claim 34 wherein the CD spectra is unaffected when the membrane is in aqueous solution containing less than about 10% sodium dodecyl sulfate, less than 7 M guanidine hydrochloride, or less than 8 M urea. 36. The membrane of claim 1 wherein the membrane is not degraded by trypsin, .alpha.-chymotrypsin, papain, protease K or pronase. 37. The membrane of claim 1 wherein the membrane is stable in serum or ethanol. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND Macroscopic membranes play an important role in many biological processes at both the cellular and organismic level. In addition, membranes are used in a number of medical, research, and industrial applications. Physiologically compatible membranes would be especially valuable for biomedical products. At present, the self-assembly of peptides into macroscopic membranes has not been reported. SUMMARY OF THE INVENTION A small peptide termed EAK16 (AEAEAKAKAEAEAKAK310-325 of SEQ.ID NO:2) was discovered serendipitously to self-assemble into stable macroscopic membranes in the presence of millimolar concentrations of salt. This invention relates to the self-assembly of peptides into stable macroscopic membranes. Peptides which form membranes are characterized as being amphiphilic, i.e., having alternating hydrophobic and hydrophilic amino acid residues; greater than 12 amino acids, and preferably at least 16 amino acids; complementary and structurally compatible. Complementary refers to the ability of the peptides to interact through ionized pairs and/or hydrogen bonds which form between their hydrophilic side-chains, and structurally compatible refers to the ability of complementary peptides to maintain a constant distance between their peptide backbones. Peptides having these properties participate in intermolecular interactions which result in the formation and stabilization of .beta.-sheets at the secondary structure level and interwoven filaments at the tertiary structure level. Both homogeneous and heterogeneous mixtures of peptides characterized by the above-mentioned properties can form stable macroscopic membranes. Peptides which are self-complementary and self-compatible can form membranes in a homogeneous mixture. Heterogeneous peptides, including those which cannot form membranes in homogeneous solutions, which are complementary and/or structurally compatible with each other can also self-assemble into macroscopic membranes. Peptides which can self-assemble into macroscopic membranes, the conditions under which membrane formation occurs, and methods for producing the membranes are described and included in this invention. Macroscopic membranes formed of the peptide EAK16 have been found to be stable in aqueous solution, in serum, and in ethanol and are highly resistant to degradation by heat, alkaline and acidic pH (i.e., stable at pH 1.5-11), chemical denaturants (e.g., guanidine-HCl, urea and sodium dodecyl sulfate), and proteases (e.g., trypsin, .alpha.-chymotrypsin, papain, protease K, and pronase). The membranes have also been found to be non-cytotoxic. The membranes are thin, transparent and resemble high density felt under high magnification. Being composed primarily of protein, the membranes can be digested and metabolized in animals and people. They have a simple composition, are permeable, and are easy and relatively inexpensive to produce in large quantities. The membranes can also be produced and stored in a sterile condition. Thus, the macroscopic membranes provided by this invention are potentially useful as biomaterial for medical products, as vehicles for slow-diffusion drug delivery, as separation matrices, and for other uses requiring permeable and water-insoluble material. Furthermore, the salt-induced assembly of the peptides into insoluble and protease-resistant protein filaments with a .beta.-sheet secondary structure is similar in some respects to the formation of the neurofibrillary filaments and amyloid plaques associated with Alzheimer's disease and the formation of scrapie prion protein filaments. The formation of the macroscopic membranes can therefore be useful as a model system to study these pathological processes. For example, such a model system can be used to identify drugs which inhibit filament formation and are thus potentially useful for treating Alzheimer's disease and scrapie infection. Peptide EAK16 was derived from a region of a yeast protein, zuotin, which exhibits a high affinity for DNA in the left-handed Z conformation. Zuotin was identified by a gel shift assay for Z-DNA binding proteins developed by the Applicants. Applicants further cloned and sequenced the gene encoding zuotin. Characterization of zuotin revealed that the protein is a potential substrate for several protein kinases and identified a putative DNA-binding domain. This invention also includes all or biologically active portions of the zuotin protein and DNA encoding zuotin. |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
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