Optimal interstrand bridge for collagen mimics
Novel collagen-mimetic peptides are disclosed comprising the formula (Xaa-Yaa-Gly) n , where the amino acid at one of the Xaa positions is substituted with a homocysteine residue. Also disclosed are multi-stranded novel collagen-mimetic peptides comprising a first strand as described above that is covalently bonded with a disulfide bridge to a second strand comprising the formula (Xaa-Yaa-Gly) m , where the amino acid at one of the Yaa positions is substituted with a cysteine residue. Disulfide formation between the terminal thiol sulfur of the homocysteine residue of the first strand and the terminal thiol sulfur of the cysteine residue of the second strand reveals unstrained bridges that enhance the structure and substantially improve the stability of collagen triple helices as compared to other possible disulfide or thioether bridges. Thus, the disclosed collagen mimetic peptides have improved stability, and can be used to produce optimized collagen-like fibrillar assemblies for wound healing and other biomedical applications.
1. A multi-stranded collagen-mimetic peptide comprising a two-stranded self-assembly unit comprising:
(a) a first strand consisting of the formula (Xaa-Yaa-Gly) n -Hcy-Yaa-Gly-(Xaa-Yaa-Gly) m or (Xaa-Yaa-Gly) n -Cys-Yaa-Gly-(Xaa-Yaa-Gly) m , wherein Gly is glycine, Hcy is homocysteine, each Xaa and each Yaa is independently any amino acid residue, and m is an integer from 1-16 and n is an integer from 1-16;
(b) a second strand consisting of the formula (Xaa-Yaa-Gly) j -Xaa-Cys-Gly-(Xaa-Yaa-Gly) k , wherein Gly is glycine, Cys is cysteine, each Xaa and each Yaa is independently any amino acid residue, and j is an integer from 1-16 and k is an integer from 1-16;
wherein the first and second strands are covalently bonded with a disulfide bond between the sulfur atom of the thiol side chain of the homocysteine residue or the cysteine residue of the first strand and the sulfur atom of the thiol side chain of the cysteine residue of the second strand to form two-stranded unit capable of associating into a triple helix.
2. The multi-stranded collagen-mimetic peptide of claim 1 , wherein each Xaa and each Yaa is independently selected from the group consisting of proline, hydroxyproline, and an amino acid residue having a side chain capable of being functionalized.
3. The multi-stranded collagen-mimetic peptide of claim 2 , wherein the residue having a side chain capable of being functionalized is lysine.
4. The multi-stranded collagen-mimetic peptide of claim 1 , wherein each Xaa is proline and each Yaa is hydroxyproline or proline.
5. A multimer formed by self-assembling the multi-stranded collagen-mimetic peptide of claim 1 into a triple helix.
6. The multi-stranded collagen-mimetic peptide of claim 1 , wherein the first strand is SEQ ID NO:8 and the second strand is SEQ ID NO:9.
7. A synthetic collagen-like fibrillar assembly comprising a plurality of the multi-stranded collagen-mimetic peptides of claim 1 .
8. A composition comprising the synthetic collagen-like fibrillar assembly of claim 7 and a therapeutic agent.
9. A kit comprising a plurality of the multi-stranded collagen-mimetic peptides of claim 1 .
10. The kit of claim 9 , further comprising a therapeutic agent.
11. A method of making a multi-stranded collagen-mimetic peptide comprising:
forming a two-stranded self-assembly unit comprising a covalent disulfide bond between:
(a) a first strand consisting of the formula (Xaa-Yaa-Gly) n -Hcy-Yaa-Gly-(Xaa-Yaa-Gly) m or (Xaa-Yaa-Gly) n -Cys-Yaa-Gly-(Xaa-Yaa-Gly) m , wherein Gly is glycine, Hcy is homocysteine, each Xaa and each Yaa is independently any amino acid residue, and m is an integer from 1-16 and n is an integer from 1-16; and
(b) a second strand consisting of the formula (Xaa-Yaa-Gly)j-Xaa-Cys-Gly-(Xaa-Yaa-Gly)k, wherein Gly is glycine, Cys is cysteine, each Xaa and each Yaa is independently selected from any amino acid, and j is an integer from 1-16 and k is an integer from 1-16;
wherein the covalent disulfide bond is formed between the sulfur atom of the thiol side chain of the homocysteine residue or the cysteine residue of the first strand and the sulfur atom of the thiol side chain of the cysteine residue of the second strand to form the two-stranded unit capable of associating into a triple helix.
12. The method of claim 11 , wherein each Xaa is proline and each Yaa is proline or hydroxyproline.
13. The method of claim 11 , further comprising self-assembling the multi-stranded collagen-mimetic peptide into a triple helix.
14. The method of claim 13 , wherein each Xaa and each Yaa is independently selected from the group consisting of proline, hydroxyproline, and an amino acid residue having a side chain capable of being functionalized.
15. A method of making a synthetic collagen-like fibrillar assembly, comprising contacting a first multi-stranded collagen-mimetic peptide of claim 1 with one or more second multi-stranded collagen-mimetic peptides of claim 1 , whereby the first and second multi-stranded collagen-mimetic peptides self-assemble into a larger synthetic collagen-like fibrillar assembly.
16. A method of facilitating wound healing, comprising contacting a wound with a composition comprising the multi-stranded collagen-mimetic peptide of claim 1 , thereby facilitating the healing of the wound.
17. The method of claim 16 , wherein the composition further comprises a therapeutic agent.
18. The multi-stranded collagen mimetic peptide of claim 1 , wherein n is 5 and m is 2.
19. The multi-stranded collagen mimetic peptide of claim 1 , wherein both j and k are independently selected from integers selected from 1 to 16.
20. The multi-stranded collagen mimetic peptide of claim 1 , wherein j is 2 and k is 5.
21. The multi-stranded collagen mimetic peptide of claim 1 , wherein each Xaa is proline and each Yaa is 4(R)-hydroxyproline (Hyp).
22. The multi-stranded collagen mimetic peptide of claim 1 , wherein the first strand is (Pro-Hyp-Gly) 5 -Hcy-Hyp-Gly-(Pro-Hyp-Gly) 2 , SEQ ID NO:8, and the second strand is (Pro-Hyp-Gly) 2 -Pro-Cys-Gly-(Pro-Hyp-Gly) 5 , SEQ ID NO:9, wherein Pro is proline, Hyp is 4(R)-hydroxyproline, Gly is glycine, Hcy is homocysteine, and Cys is cysteine.
23. The method of claim 11 , wherein the first strand is SEQ ID NO:8 and the second strand SEQ ID NO:9.
24. A synthetic collagen-like fibrillar assembly made by the method of claim 15 .
25. A method of facilitating wound healing, comprising contacting a wound with a composition comprising the synthetic collagen-like fibrillary assembly of claim 24 , thereby facilitating the healing of the wound.