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 ...

Targeting collagen strands by photo-triggered triple-helix hybridization

“…The results confirm that CMP's binding affinity to intact type I collagen is real and also stereo-selective. We speculate that the low-level binding is caused by CMPs hybridizing with thermally unstable domains of the native collagen (19) or collagens partially denatured during purification and fiber regeneration. The density of photo-induced CMP binding to intact collagen was determined to be as high as 0.56 AE 0.03 nmol∕cm 2 , which is well above the bioactive ligand density for a variety of cell scaffold interactions in cell culture and tissue development (20,21).…”

Spatio-Temporal Modification of Collagen Scaffolds Mediated by Triple Helical Propensity

“…We previously reported the poly(proline)-II-like structure of single-stranded CMPs anchored to the surfaces of these gold nanoparticles, as well as the NPs' high colloidal stability in a wide range of salt and pH conditions and their binding affinities to type I bovine collagen fibers. 18 In our current study, we tested type I collagen isolated from mouse tails, which is known to form higher quality collagen fibers with more prominent banding features compared to bovine collagen previously tested. 26 Room temperature incubation of these collagen fibers with gold NPs displaying Cys-(ProHypGly) 7 (CysCMP-7, Table 1) produced intact fibers decorated with a large number of NPs similar to those observed for bovine type I collagen.…”

“…11,16 Synthetic collagen model systems, also known as collagen mimetic peptides (CMPs), have been useful in elucidating collagen structure and factors responsible for the stabilization of the triple helix. 9,[17][18][19] CMPs form a triple helical structure almost identical to that of natural collagens; however, unlike collagen molecules, CMPs are small (<3 KDa) and, therefore, exhibit reversible melting behaviors. 11,13,17 CMPs based on ProProGly or ProHypGly trimers have been widely studied and their melting temperatures (T m ) vary from 21 to 75 °C, depending on the molecular weight and amino acid composition.…”

“…11,13,17 CMPs based on ProProGly or ProHypGly trimers have been widely studied and their melting temperatures (T m ) vary from 21 to 75 °C, depending on the molecular weight and amino acid composition. 9,[11][12][13][18][19][20] Considering the rare occurrence of triple helical protein structure in nature outside of collagen, we thought that the associative interactions between collagen strands in forming the triple helix can be explored as a unique physical conjugation tool for attaching exogenous molecules to natural collagens. Physical interactions, such as hydrogen bonding, hydrophobic, and ionic interactions and biological affinity interactions, provide a much more convenient and natural way of conjugating agents to proteins and other macromolecules as opposed to covalent chemical attachment.…”

Preparation of gold nanoparticle/single-walled carbon nanotube nanohybrids using biologically programmed peptide for application of flexible transparent conducting films

“…Biomolecules such as DNA, peptides, and proteins are particularly promising candidate biotemplates and structure-guiding components for the synthesis and assembly of nanomaterials and their integration into organic or inorganic materials [5]. The development of metal binding peptides using genetic engineering approaches has resulted in several biological strategies to tailor nanoparticle (NP) physicochemical properties and to assembly NPs at multiple length scales by utilizing the sequence programmability, selective molecular recognition ability, and multi-functionality of metal binding peptides [6][7][8][9]. In addition, fusion peptides with two domains have been used to synthesize bimetallic NPs [10].…”