Controlling collagen triple helix formation and higher order assembly

Jee Yeon Lee, Purdue University

Abstract

Collagen is one of the most abundant groups of proteins that constitute the major components of the extracellular matrix, connective tissues, ligaments, tendons, skin, bone and cartilage with high complexity, diversity, organization and function. The ability to control triple helix formation or higher order assemblies of collagen peptides would be useful for biomaterial applications. We have engineered a pH-responsive CTH peptide that includes modified hydroxyproline residues with a carboxylate moiety (P E) along the collagen peptide. The introduction of one or three P E residues into a collagen peptide had little effect on triple helix stability at acidic and neutral pH. However, the introduction of five or seven PE residues within a collagen peptide demonstrated that a CTH may form under acidic conditions, but not at neutral pH. These data support a pH dependent CTH formation. Furthermore, we used the PE residues to perturb the folding in localized sites along the collagen peptide due to the negative charges. We demonstrated that the addition of PE residues into collagen peptides affects the folding of the CTH, as well as the thermal stability. Increasing the numbers of charged PE residues within the peptide was found to reduce the refolding rate of CTH formation due to electrostatic repulsion. Moreover, the specific placement of PE residues within the collagen peptide allowed us to selectively test for possible terminal and central nucleation. These studies indicate that incorporation of PE residues may be a strategy to study the folding mechanism of collagen peptides. Our strategy was further expanded to promote the higher order assembly of collagen triple helices by electrostatic interactions from charges found on the modified hydroxyproline residues. The negative and positive charges were introduced into a (Pro-Hyp-Gly)9 scaffold through modification of the hydroxyproline residues with carboxylate (PE) and amine (PK) functionality by O-alkylation. Combination of the two triple helical collagen peptides, the negatively charged 3/3E/3 and the positively charged 3/3K/3, was found to facilitate radial growth of collagen structures via electrostatic interactions into disk shaped nanostructures. This study suggests that the higher order assembly of collagen triple helices could be promoted by electrostatic interactions with varied environmental conditions resulting in a different morphology for the assembled structure with a different growth mechanism. Our strategy to control the supramolecular assembly of collagen peptides was further extended to use of hydrophobic interactions. Incorporation of a hydrophobically-modified hydroxyproline residue (PL) into collagen peptides allowed us to observe different assembly characteristics depending on the position of the hydrophobic PPLG units in the peptide. The PPLG-4T peptide, containing PPLG units at the C- and N-termini of the peptide, was found to form heterotrimers with a neutral and shorter POG-7 peptide through a heat-triggered or self-triggered mechanism. The hydrophobic sticky ends generated by the formation of heterotrimers offer the possibility for head to tail assembly. The PPLG-4G peptide, containing hydrophobic segments embedded within a (Pro-Hyp-Gly) 10 peptide, was able to form round assemblies suggesting that it may grow in radial direction via hydrophobic interactions. These results suggest that functionalizing collagen peptides by alternating the position of the PL residues may trigger the peptide to perform strand-invasion or self-assembly. Metal coordination has been a powerful strategy to promote higher order assemblies in collagen model peptides. We incorporated Gla (γ-carboxyglutamic acid) residues into a collagen peptide to investigate the metal-promoted higher order assembly of CTH. The Gla-1 peptide, containing a central Gla residue, was able to form assembled structures with various metals, including LaCl3, FeCl3 and GdCl3. Metal-assisted assembled micro- or nano-structures of Gla-1 were found with a variety of morphologies and sizes. Our various strategies for controlling triple helical conformation or higher order assemblies of collagen peptides could provide the basis for new biomaterial applications.

Degree

Ph.D.

Advisors

Chmielewski, Purdue University.

Subject Area

Organic chemistry|Polymer chemistry

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