Polypeptide compositions formed using a coiled-coil template and methods of use
View Patent ↗The invention relates to the use of constrained coiled-coil polypeptides to mimic α-helical structural elements of native proteins. These constrained peptidyl mimetics are used to generate and/or identify ligands, which selectively bind the α-helical segment contained in the native protein.
1. A coiled-coil polypeptide comprising the formula (ab i c i de i f i g i ) n , where i=1, 2, . . . , n, and n is at least three, said polypeptide being prepared by
(a) selecting a solvent-accessible region of an epitope of a selected natural protein, wherein said region is not in a coiled-coil conformation in its native state, and inserting the amino acids from said region into the b i , c i , e i , f i and g i positions; and
(b) independently inserting an amino acid selected from the group consisting of leucine, isoleucine, valine, phenylalanine, methionine, tyrosine, and derivatives thereof, into each of the a and d positions such that the amino acids from the epitope in the b i , c i , e i , f i and g i positions are interrupted by the amino acids in the a and d positions;
wherein (ab i c i de i f i g i ) n , forms a coiled-coil.
2. The polypeptide of claim 1 , wherein a is isoleucine and d is leucine.
3. The polypeptide of claim 1 , wherein the coiled-coil polypeptide is comprised of two polypeptide chains arranged in a parallel configuration.
4. The polypeptide of claim 1 , wherein n is between about 3 and about 20.
5. The polypeptide of claim 1 , wherein the epitopes are selected from α-helical surface regions of a cellular prion protein.
6. The polypeptide of claim 5 , wherein the epitopes are selected from exposed surface regions of an infectious prion protein.
7. The polypeptide of claim 5 , wherein the sequence formed by the positions (b i c i e i f i g i ) n corresponds to the solvent-accessible residues of an epitope having a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
8. The polypeptide of claim 5 , wherein the cellular prion protein is selected from the group consisting of mouse, hamster, bovine, ovine and human cellular prion proteins.
9. A coiled-coil polypeptide, comprising an amino acid sequence represented by (ab i c i de i f i g i ) n , where
i=1, 2, . . . , n, and n is at least three;
a and d are amino acids each independently selected from the group consisting of leucine, isoleucine, valine, phenylalanine, methionine, tyrosine and derivatives thereof;
(b i c i e i f i g i ) n is a sequence of amino acids from a solvent-accessible region of an epitope from a selected natural protein, wherein said region is not in a coiled-coil conformation in its native state, and the sequence (b i c i e i f i g i ) n is interrupted by the amino acids in the a and d positions in (ab i c i de i f i g i ) n ; and
wherein (ab i c i de i f i g i ) n forms a coiled coil.
10. The polypeptide of claim 9 , wherein a is isoleucine and d is leucine.
11. The polypeptide of claim 9 , wherein the coiled-coil polypeptide is comprised of two polypeptide chains arranged in a parallel configuration.
12. The polypeptide of claim 9 , wherein n is between about 3 and about 20.
13. The polypeptide of claim 9 , wherein the epitopes are selected from α-helical surface regions of a cellular prion protein.
14. The polypeptide of claim 9 , wherein the epitopes are selected from exposed surface regions of an infectious prion protein.
15. The polypeptide of claim 13 , wherein the sequence formed by the positions (b i c i e i f i g i ) n corresponds to the solvent-accessible residues of an epitope having a sequence selected from the group consisting of SEQ ID NO: 5, SEQ D NO: 6, and SEQ ID NO: 7.
16. The polypeptide of claim 13 , wherein the cellular prion protein is selected from the group consisting of mouse, hamster, bovine, ovine and human cellular prion proteins.
17. A coiled-coil polypeptide comprising the formula (ab i c i de i f i g i ), where i=1, 2 . . . , n, and n is between about 5 and about 10, said polypeptide being prepared by
(a) selecting a solvent-accessible region of an epitope of a selected natural protein, wherein said region is not in a coiled-coil conformation in its native state, and inserting the amino acids from said region into the b i , c i , e i , f i and g i positions; and
(b) independently inserting an amino acid selected from the group consisting of leucine, isoleucine, valine, phenylalanine, methionine, tyrosine, and derivatives thereof, into each of the a and d positions such that the amino acids from the epitope in the b i , c i , e i , f i and g i positions are interrupted by the amino acids in the a and d positions;
wherein (ab i c i de i f i g i ) n , forms a coiled-coil.
18. The polypeptide of claim 17 , wherein a is isoleucine and d is leucine.
19. The polypeptide of claim 17 , wherein the coiled-coil polypeptide is comprised of two polypeptide chains arranged in a parallel configuration.
20. The polypeptide of claim 17 , wherein the epitopes are selected from α-helical surface regions of a cellular prion protein.
21. The polypeptide of claim 17 , wherein the epitopes are selected from exposed surface regions of an infectious prion protein.
22. The polypeptide of claim 20 , wherein the sequence formed by the positions (b i c i e i f i g i ) n corresponds to the solvent-accessible residues of an epitope having a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
23. The polypeptide of claim 20 , wherein the cellular prion protein is selected from the group consisting of mouse, hamster, bovine, ovine and human cellular prion proteins.
24. A coiled-coil polypeptide, comprising an amino acid sequence represented by (ab i c i de i f i g i ) n , where i=1, 2, . . . , n, and n is between about 5 and about 10;
a and d are amino acids each independently selected from the group consisting of leucine, isoleucine, valine, phenylalanine, methionine, tyrosine and derivatives thereof;
(b i c i e i f i g i ) n is a sequence of amino acids from a solvent-accessible region of an epitope from a selected natural protein, wherein said region is not in a coiled-coil conformation in its native state, and the sequence (b i c i e i f i g i ) n is interrupted by the amino acids in the a and d positions in (ab i c i de i f i g i ) n ; and
wherein (ab i c i de i f i g i ) n forms a coiled coil.
25. The polypeptide of claim 24 , wherein a is isoleucine and d is leucine.
26. The polypeptide of claim 24 , wherein the coiled-coil polypeptide is comprised of two polypeptide chains arranged in a parallel configuration.
27. The polypeptide of claim 24 , wherein the epitopes are selected from α-helical surface regions of a cellular prion protein.
28. The polypeptide of claim 24 , wherein the epitopes are selected from exposed surface regions of an infectious prion protein.
29. The polypeptide of claim 27 , wherein the sequence formed by the positions (b i c i e i f i g i ) corresponds to the solvent-accessible residues of an epitope having a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
30. The polypeptide of claim 27 , wherein the cellular prion protein is selected from the group consisting of mouse, hamster, bovine, ovine and human cellular prion proteins.