Collagen is a ubiquitous biomaterial that forms the supporting structures in skin, bone, tendons, cartilage, and blood vessels. Numerous types of collagen have been identified, and the tertiary structure of each shares the common structural motif of the collagen triple helix (CTH). [1] The CTH motif is composed of three chains, each of which adopts a left-handed type-II polyproline helix, that come together to form a righthanded superhelix. [2] Repeating units of GlyXaaYaa are common within the different types of collagen, and a tendency for X to be proline and Y to be hydroxyproline (Hyp, O) has been observed. [3] Minimal peptide sequences based on this idealized sequence have provided a wealth of information concerning the structural and sequence requirements for triple-helix stability. [4] The ability to control triplehelix formation fundamentally would be useful for a range of collagen-based biomaterial applications, such as tissue engineering and drug delivery. [5] Herein we disclose modifications to collagen peptides that lead to the formation of triple helices on demand through environmental control.
Our plan for the design of a pH-responsive CTH was to include carboxylate moieties along the collagen peptide. [6] Under neutral conditions, interstrand electrostatic repulsion would disfavor triple-helix formation. However, when the carboxylate groups are protonated under acidic conditions, a stable triple helix should form, as long as steric repulsion from the appended groups is not a factor. This design is complementary to the use of electrostatic interactions to promote the stabilization of CTHs. [7] As we wished to minimize alteration to the (POG) n helical structure, our design strategy was to incorporate a carboxylate functionality within the Hyp residue by O-alkylation [8] (to give a P E residue; Figure 1). Molecular modeling of a CTH with the inclusion of P E suggested that this nonnatural amino acid should be accommodated well with minimal interstrand steric interactions (Figure 1 a).
Five peptides, therefore, formed the basis of this study: two control peptides containing seven repeating units of POG or PEG (POG-7 and PEG-7, respectively), two host-guest peptides containing one or three central PP E G units (PP E G-1 and PP E G-3, respectively), and PP E G-7, which contains seven repeating units of PP E G (Figure 1 b). The host-guest peptide