Nanoparticle‐Assisted Visualization of Binding Interactions between Collagen Mimetic Peptide and Collagen Fibers

Abstract

Collagen is the structural framework for a wide range of animal connective tissues. It is known to induce cell proliferation and differentiation by direct epitope binding and by serving as a reservoir for growth factors and signaling molecules. [1] In the past, collagen mimetic peptide (CMP), a synthetic peptide composed of a collagen-like repetitive amino acid sequence, has played a central role in elucidating the triple-helical structure and thermal stability of natural collagens. [2][3][4][5][6][7] Herein, we show for the first time that a biochemically inert but highly helicogenic CMP, (Pro-HypGly) 7 (Hyp = hydroxyproline), binds preferentially to the gap regions within the surface of intact type I collagen fibers. This binding behavior was demonstrated by synthesizing CMPconjugated gold nanoparticles (NPs) that are colloidally stable under a wide range of aqueous conditions and by transmission electron microscopy (TEM) observation of their attraction to type I collagen fibers under physiological conditions. The results suggest that such binding affinity may exist in other natural proteins/peptides that contain collagen-like sequences.[8] The study also demonstrates the potential use of nanoparticle-labeling techniques in detecting structurally unstable domains in collagen fibers which are related to many debilitating human diseases. [9] The collagen triple helix is composed of three poly-(proline)-II strands that are held together by interchain hydrogen bonds. [4,10] This structure is similar to the doublehelical structure of DNA. Both collagen and DNA exhibit melting transitions that reflect the stability and strength of the multiplex assemblies. Strand invasion in DNA by short DNA strands or peptide nucleic acids is well documented. [11] Although collagens are known to incorporate thermally unstable domains where small segments of the triple helix are thought to be partially unraveled, [12] strand invasion by other collagen molecules or collagen analogues have not been reported to date.We previously presented a method of collagen modification that uses CMP as a collagen-specific molecular "hitchhiker".[13] Our results suggested that the CMP, (Pro-HypGly) x binds to a film composed of type I collagen molecules (in nonfibrous form) through a process that involves both strand invasion and assembly of a triple helix. However, the binding location and physical state of the collagen molecules that interact with CMP have not been studied in detail.In nature, type I collagen exists not as individual molecules but instead forms fibrous bundles of high tensile strength. Under TEM, collagen fibers exhibit characteristic banding patterns that indicate the structural integrity of the collagen molecules and the regularity of their assembly. The banding patterns also provide approximate position markers along the length of collagen molecules. As CMP is invisible under TEM, we prepared CMP-conjugated gold nanoparticles to use as an electron-dense TEM marker for the location of CMPs. We investigated binding between type I ...

Enhanced Chondrogenesis of Mesenchymal Stem Cells in Collagen Mimetic Peptide-Mediated Microenvironment

“…15,16 The binding affinity originates from CMP's strong propensity to form a triple helix. It is believed that the observed adhesion between CMP and collagen arises from a single-stranded CMP invading structurally unstable domains of collagen molecule and forming a stable product secured by a triple helix assembly.…”

“…Collagen mimetic peptides (CMPs), -(Pro-Hyp-Gly) 7 -(Hyp ¼ hydroxyproline), have a unique collagen-like triple helical conformation and have been shown to associate with collagen molecules and fibers via a strand invasion process. 15,16 As an effort to develop a bioactive 3D PEODA hydrogel suitable for encapsulated cells, we produced a PEODA scaffold conjugated with CMP (CMP/PEODA). 17 In this study, we demonstrate that CMP/PEODA provides a suitable bio-active microenvironment, facilitating the efficient chondrogenic differentiation of MSCs.…”

The non-covalent decoration of self-assembling protein fibers

“…In this vein, Yu's group has functionalized collagen-based scaffolds with Collagen Mimetic Peptides (CMPs) [16,17]; and has employed anionic CMPs to attach cell-growth factors to collagen scaffolds [18]. Coulombic interactions have also been used to organize gold nanoparticles and coiled-coils into reversible systems [19].…”

Peptides and Metallic Nanoparticles for Biomedical Applications

“…(D) There was no such preference when the collagen fibers and NP-3 were incubated at 40°C and cooled to room temperature. Reproduced with permission from [74]. Biomedical applications of peptide-Co NP conjugates Thanh and colleagues were the first to report the use of peptides as capping ligands for in situ synthesis of water-soluble CoNPs with the aim to apply these particles for biological purposes [83].…”

“…Mo and colleagues demonstrated the potential of NP-labeling techniques in detecting structurally unstable domains in collagen fibers, which are related to many debilitating human diseases [74]. They showed for the first time that a biochemically inert but highly helicogenic cartilage matrix protein (CMP; Pro-Hyp-Gly) 7 (Hyp = hydroxyproline), binds preferentially to the gap regions on the surface of intact type I collagen fibers.…”