Peptoid Residues Make Diverse, Hyperstable Collagen Triple-Helices.

As the only ribosomally encoded N-substituted amino acid, proline promotes distinct secondary protein structures. The high proline content in collagen, the most abundant protein in the human body, is crucial to forming its hallmark structure: the triple-helix. For over five decades, proline has been considered compulsory for synthetic designs aimed at recapitulating collagen's structure and properties. Here we describe that N-substituted glycines (N-glys), also known as peptoid residues, exhibit a general triple-helical propensity similar to or greater than proline, enabling synthesis of stable triple-helical collagen mimetic peptides (CMPs) with unprecedented side chain diversity. Supported by atomic-resolution crystal structures as well as circular dichroism and computational characterizations spanning over 30 N-gly-containing CMPs, we discovered that N-glys stabilize the triple-helix primarily by sterically preorganizing individual chains into the polyproline-II helix. We demonstrated that N-glys with exotic side chains including a "click"-able alkyne and a photosensitive side chain enable CMPs for functional applications including the spatiotemporal control of cell adhesion and migration. The structural principles uncovered in this study open up opportunities for a new generation of collagen-mimetic therapeutics and materials.

Insertion of Pro-Hyp-Gly provides 2 kcal mol-1 stability but attenuates the specific assembly of ABC heterotrimeric collagen triple helices.

Collagen is a major structural component of the extracellular matrix and connective tissue. The key structural feature of collagen is the collagen triple helix, with a Xaa-Yaa-Gly (glycine) repeating pattern. The most frequently occurring triplet is Pro (proline)-Hyp (hydroxyproline)-Gly. The reversible thermal folding and unfolding of a series of heterotrimeric collagen triple helices with varying number of Pro-Hyp-Gly triplets were monitored by circular dichroism spectroscopy to determine the unfolding thermodynamic parameters Tm (midpoint transition temperature), ΔHTm (unfolding enthalpy), and ΔGunfold (unfolding free energy). The Tm and ΔGunfold of the heterotrimeric collagen triple helices increased with increasing number of Pro-Hyp-Gly triplets. The ΔGunfold increased by 2.0 ± 0.2 kcal mol-1 upon inserting one Pro-Hyp-Gly triplet into all three chains. The Tm difference between the most stable ABC combination and the second most stable BCC combination decreased with increasing number of Pro-Hyp-Gly triplets, even though the ΔGunfold difference remained the same. These results should be useful for tuning the stability of collagen triple helical peptides for hydrogel formation, recognition of denatured collagen triple helices as diagnostics and therapeutics, and targeted drug delivery.

Bioengineered Collagens.

There is a great deal of interest in obtaining recombinant collagen as an alternative source of material for biomedical applications and as an approach for obtaining basic structural and biological information. However, application of recombinant technology to collagen presents challenges, most notably the need for post-translational hydroxylation of prolines for triple-helix stability. Full length recombinant human collagens have been successfully expressed in cell lines, yeast, and several plant systems, while collagen fragments have been expressed in E. coli. In addition, bacterial collagen-like proteins can be expressed in high yields in E. coli and easily manipulated to incorporate biologically active sequences from human collagens. These expression systems allow manipulation of biologically active sequences within collagen, which has furthered our understanding of the relationships between collagen sequences, structure and function. Here, recombinant studies on collagen interactions with cell receptors, extracellular matrix proteins, and matrix metalloproteinases are reviewed, and discussed in terms of their potential biomaterial and biomedical applications.

Synthesis and Fabrication of Nanocomposite Fibers of Collagen-Cellulose Nanocrystals by Coelectrocompaction.

An electrochemical process has been used to compact cellulose nanocrystals (CNC) and access aligned micron-sized CNC fibers. Placing a current across aqueous solutions of carboxylic acid functionalized CNCs (t-CNC-COOH) or carboxylic acid/primary amine functionalized CNCs (t-CNC-COOH-NH2) creates a pH gradient between the electrodes, which results in the migration and concentration of the CNC fibers at their isoelectric point. By matching the carboxylic acid/amine ratio of CNCs and collagen (ca. 30:70 carboxylic acid:amine ratio), it is possible to coelectrocompact both nanofibers and access aligned nanocomposite fibers. t-CNC-COOH-NH2/collagen fibers showed a maximum increase in mechanical properties at 5 wt % of t-CNC-COOH-NH2. Compared to collagen/CNC films which have no alignment in the plane of the films, the tensile properties of the aligned fibers show a significant enhancement in the wet mechanical properties (40 MPa vs 230 MPa) for the 5 wt % of t-CNC-COOH-NH2/collagen films and fiber, respectively.

Reversible Temperature-Switching of Hydrogel Stiffness of Coassembled, Silk-Collagen-Like Hydrogels.

Recombinant protein polymers, which can combine different bioinspired self-assembly motifs in a well-defined block sequence, have large potential as building blocks for making complex, hierarchically structured materials. In this paper we demonstrate the stepwise formation of thermosensitive hydrogels by combination of two distinct, orthogonal self-assembly mechanisms. In the first step, fibers are coassembled from two recombinant protein polymers: (a) a symmetric silk-like block copolymer consisting of a central silk-like block flanked by two soluble random coil blocks and (b) an asymmetric silk-collagen-like block copolymer consisting of a central random-coil block flanked on one side by a silk-like block and on the other side a collagen-like block. In the second step, induced by cooling, the collagen-like blocks form triple helices and thereby cross-link the fibers, leading to hydrogels with a thermo-reversibly switchable stiffness. Our work demonstrates how complex self-assembled materials can be formed through careful control of the self-assembly pathway.

Synthesis and characterization of monodisperse poly(ethylene glycol)-conjugated collagen pentapeptides with collagen biosynthesis-stimulating activity.

Although collagen pentapeptide (Lys-Thr-Thr-Lys-Ser, KTTKS) has received a great deal of attention owing to its collagen biosynthesis-stimulating effects, its enzymatic instability in the skin is an obstacle to effective topical application. PEGylation is a useful approach for improving the chemical and biological stability of peptides. However, the polydispersity of poly(ethylene glycol) (PEG) produces conjugates with different molecular sizes, which may create difficulties in chemical characterization and purity control, and in variability of biological properties. To overcome these difficulties, monodisperse PEG was site-specifically conjugated to the N-terminal amine of KTTKS to produce a single molecular conjugate, enabling more complete chemical characterization and more exact product specifications. PEG-KTTKS conjugates prepared using monodisperse PEG with two different molecular weights, monodisperse PEG220 and PEG572, were characterized by mass spectrometry. These monodisperse PEG-KTTKS conjugates showed no cytotoxicity (1-100 µM) and stimulated collagen biosynthesis in human skin fibroblasts. They also had high stability against proteolytic enzymes in rat skin. This study demonstrates the usefulness of monodisperse PEG for preparing chemically defined conjugates and suggests that monodisperse PEG-KTTKS would be a good candidate for use as a collagen biosynthesis-stimulating agent.

Production of collagen micro- and nanofibers for potential drug-carrier systems.

Two different nano- and micro-collagen fiber production methods are introduced and discussed. First one is the electrospinning method, that is very common technique to produce nanofibers from different polymeric solutions and recently collagen solutions are employed to produce nanofibers for different biomedical applications. This technique is extremely versatile method to produce nanofibers in a relatively short time, easy to control the fiber diameter and orientation with small pore sizes and a high surface area. The second method is self-assembly of collagen micro-fibers by co-extrusion method. The collagen fibers are obtained without any cross-linker, by using mainly ionic interactions. We demonstrated that self-assembled collagen fibers have well preserved their native structure (0.90 PP-II fraction), when compared with electrospun collagen fibers (0.38 PP-II fraction). However, it was only possible to produce collagen fibers with nanodimensions by using electrospinning method.

Synthesis and characterization of collagen grafted poly(hydroxybutyrate-valerate) (PHBV) scaffold for loading of bovine serum albumin capped silver (Ag/BSA) nanoparticles in the potential use of tissue engineering application.

The objective of this study is to synthesize and characterize collagen grafted poly(3-hydroxylbutyrate-co-3-hydroxylvalerate) (PHBV) film for loading of BSA capped silver (Ag/BSA) nanoparticles. Thermal radical copolymerization and aminolysis methods were used to functionalize macroporous PHBV, followed by collagen grafting so as to formulate collagen-g-poly(hydroxyethylmethyl acrylate)-g-poly(3-hydroxylbutyrate-co-3-hydroxylvalerate) [collagen-g-PHEMA-g-PHBV] and collagen-g-aminated-poly(3-hydroxylbutyrate-co-3-hydroxylvalerate) [collagen-g-NH2-PHBV] films, respectively. Spectroscopic (FTIR, XPS), physical (SEM), and thermal (TGA) techniques were used to characterize the functionalized PHBV films. The amount of collagen present on grafted PHBV film was quantified by the Bradford method. The Ag/BSA nanoparticles were then loaded on collagen grafted and untreated PHBV films, and the nanoparticles loading were determined by atomic absorption spectrometry. The amount of nanoparticles loaded on collagen grafted PHBV film was found to be significantly greater than that on the untreated PHBV film. The nanoparticles loaded PHBV film can potentially serve as a scaffold to promote the growth of bone cells while inhibiting the bacterial growth.

Fabrication of solid collagen nanoparticles using electrospray deposition.

Collagen is a promising biomaterial for drug delivery due to advantages including high biocompatibility and biodegradable property. However, transforming collagen into solid nanoparticles is difficult, although the solid dosage form is advantageous for some administration routes including pulmonary and oral drug delivery. In this study, collagen solid nanoparticles are prepared in one-step using electrospray deposition under ambient temperature and pressure conditions. Although collagen molecules formed micron-sized aggregates in acetic acid solutions spontaneously, electrospraying the collagen solutions resulted in formation of nanofibers. Solid nanoparticles were obtained by increasing conductivity of the solution and/or inducing structural perturbation of the collagen molecules using salts. The ability of solid collagen particles as a drug carrier was demonstrated by incorporating theophylline as a model drug using a coaxial spray technique. Release of theophylline was controlled by cross-linking collagen molecules. Electrospray deposition was proved to be a powerful method for producing solid collagen nanoparticles for drug delivery.

One-step synthesis of collagen hybrid gold nanoparticles and formation on Egyptian-like gold-plated archaeological ivory.

A one-step method is reported to synthesize hybrid gold nanoparticles (AuNPs) by reduction of HAuCl4 in acetic solution in the presence of collagen (Col), dicarboxylic acid-terminated polyethylene glycol (PEG), and cetyltetrammonium bromide (CTAB) mixed with hydoxyapatite (HAP) as surfactants. Such formation process of AuNPs was shown to be responsible for purple stains naturally formed on Egyptianizing archaeological gilded ivories from 8th BC Syria. The understanding of this formation mechanism, which most likely involves a step with hybrid AuNPs, allows the establishing of an authenticity marker of ancient gold-plated ivories.

Structural insights into charge pair interactions in triple helical collagen-like proteins.

The collagen triple helix is the most abundant protein fold in humans. Despite its deceptively simple structure, very little is understood about its folding and fibrillization energy landscape. In this work, using a combination of x-ray crystallography and nuclear magnetic resonance spectroscopy, we carry out a detailed study of stabilizing pair-wise interactions between the positively charged lysine and the negatively charged amino acids aspartate and glutamate. We find important differences in the side chain conformation of amino acids in the crystalline and solution state. Structures from x-ray crystallography may have similarities to the densely packed triple helices of collagen fibers whereas solution NMR structures reveal the simpler interactions of isolated triple helices. In solution, two distinct types of contacts are observed: axial and lateral. Such register-specific interactions are crucial for the understanding of the registration process of collagens and the overall stability of proteins in this family. However, in the crystalline state, there is a significant rearrangement of the side chain conformation allowing for packing interactions between adjacent helices, which suggests that charged amino acids may play a dual role in collagen stabilization and folding, first at the level of triple helical assembly and second during fibril formation.

Chemical structure, biosynthesis and synthesis of free and glycosylated pyridinolines formed by cross-link of bone and synovium collagen.

This review focuses on the chemical structure, biosynthesis and synthesis of free and glycosylated pyridinolines (Pyds), fluorescent collagen cross-links, with a pyridinium salt structure. Pyds derive from the degradation of bone collagen and have attracted attention for their use as biochemical markers of bone resorption and to assess fracture risk prediction in persons suffering from osteoporosis, bone cancer and other bone or collagen diseases. We consider and critically discuss all reported syntheses of free and glycosylated Pyds evidencing an unrevised chemistry, original and of general utility, analysis of which allows us to also support a previously suggested non-enzymatic formation of Pyds in collagen better rationalizing and justifying the chemical events.

Conformational stability of triazolyl functionalized collagen triple helices.

Functionalized collagen is attractive for the development of synthetic biomaterials. Herein we present the functionalization of azidoproline containing collagen model peptides with various alkynes using click chemistry. The influence on the stability of the collagen triple helix of the stereochemistry of the introduced triazolyl prolines (4R or 4S), the position of their incorporation (Xaa or Yaa) and the substituents attached to them are shown. The results provide a useful guide for the optimal functionalization of collagen using click chemistry.

Development of a salmon-derived crosslinked atelocollagen sponge disc containing osteogenic protein-1 for articular cartilage regeneration: in vivo evaluations with rabbits.

BACKGROUND: We have developed crosslinked salmon-derived atelocollagen sponge, which has a denaturation temperature of 47 degrees Celsius. The purpose of this study is to evaluate the fundamental in vivo efficacy of the osteogenic protein (OP) -1 containing salmon-derived collagen sponge disc (SCS) on cartilage regeneration, using a rabbit model. METHODS: A total of 24 rabbits were used in this study. In each animal, a full-thickness osteochondral defect was created in each femoral trochlea. Then, each 12 rabbits were randomly divided into the two groups. In Group I, an OP1-SCS disc was implanted into the defect in the right knee. In Group II, a SCS disc without OP-1 was implanted into the defect in the right knee. A control group of 12 rabbits was assembled from randomly-selected left knees from among the first two groups. In Group-III, we applied no treatment for a defect in the left knee to obtain the untreated control. All rabbits were sacrificed at 12 weeks after surgery. In each group, 10 animals were used for histological and immunohistological evaluations, and the remaining 2 were used for real-time polymerase chain reaction (PCR) analyses. RESULTS: In Group I, a regenerated cartilage tissue rich in proteoglycan and type-2 collagen was found at 12 weeks, although the width was thicker than that of Group II. In Group II, the defect was filled with thick inhomogeneous tissues, including cartilage, fibrous, and bone tissues at 12 weeks. Concerning the gross observation and histological scores at 12 weeks, the ANOVA showed significant differences (p < 0.0001, and p < 0.0001, respectively). The post-hoc test indicated that the gross observation and histological scores of Group I was significantly greater than those of Groups II (p = 0.035, and p = 0.0104, respectively) and III (p < 0.0001, and p < 0.0001, respectively), while Group II was significantly greater than Group III (p = 0.0069, and p = 0.005, respectively). The real time PCR analysis showed that gene expression of type-2 collagen and aggrecan of Group I was greater than that of Group II. CONCLUSIONS: The present study clearly demonstrated that the implantation of the OP1-SCS disc without any cultured cells may induce spontaneous hyaline-like cartilage regeneration to greater degrees than implantation of only the salmon-derived collagen sponge disc.

Injectable collagen/α-tricalcium phosphate cement: collagen-mineral phase interactions and cell response.

A bone inspired material was obtained by incorporating collagen in the liquid phase of an α-tricalcium phosphate cement, either in solubilized or in fibrilized form. This material was able to set in situ, giving rise to a calcium deficient hydroxyapatite (CDHA)/collagen composite. The morphology and distribution of collagen in the composite was shown to be strongly affected by the collagen pre-treatment. The interactions between collagen and the inorganic phase were assessed by FTIR. A red shift of the amide I band was indicative of calcium chelation by the collagen carbonyl groups. The rate of CDHA formation was not affected when diluted collagen solutions (1 mg/ml) were used, whereas injectability improved. The presence of solubilized collagen, even in low amount (1 %), increased cell adhesion and proliferation on the composites. Still in the absence of osteogenic medium, significant ALP activity was detected both in the inorganic and the collagen-containing cements. The maximum ALP activity was advanced in the collagen-containing cement as compared to the inorganic cement.

Consequences of incorporating thiaproline and its oxidized derivatives into collagen triple helices.

(2R)-4-thiaproline (Thp) is an analog of proline, replacing Cγ in the pyrrolidine ring with sulfur. Its thiazolidine ring easily interconverts between endo and exo puckers due to a small energy barrier, which leads to destabilize polyproline helices. Collagen, composed of three polyproline II helices, mainly consists of X-Y-Gly triplets, where X is often proline and Y is frequently (2S,4R)-hydroxyproline. In this study, we incorporated Thp into either position-X or position-Y to investigate the consequences of such a replacement on the triple helix. Circular dichroism and differential scanning calorimetry analyses showed that the Thp-containing collagen-mimetic peptides (CMPs) can fold into stable triple helices, in which the substitution at position-Y exhibits a larger destabilization effect. Additionally, we also prepared the derivative peptides by oxidizing Thp in the peptide to N-formyl-cysteine or S,S-dioxide Thp. The results showed that the oxidized derivatives at position-X only slightly affect collagen stability, but those at position-Y induce a large destabilization effect. The consequences of incorporating Thp and its oxidized derivatives into CMPs are position dependent. Computational results suggested that the ease of interconversion between exo and endo puckers for Thp and the twist conformation of S,S-dioxide Thp may cause the destabilization effect at position-Y. We have revealed new insights into the impacts of Thp and its oxidized derivatives on collagen and demonstrated that Thp can be used to design collagen-related biomaterials.

Effect of sterically demanding substituents on the conformational stability of the collagen triple helix.

The effect of sterically demanding groups at proline residues on the conformational stability of the collagen triple helix was examined. The thermal stabilities (T(m) and ΔG) of eight different triple helices derived from collagen model peptides with (4R)- or (4S)-configured amidoprolines bearing either methyl or bulkier tert-butyl groups in the Xaa or Yaa position were determined and served as a relative measure for the conformational stability of the corresponding collagen triple helices. The results show that sterically demanding substituents are tolerated in the collagen triple helix when they are attached to (4R)-configured amidoprolines in the Xaa position or to (4S)-configured amidoprolines in the Yaa position. Structural studies in which the preferred conformation of (4R)- or (4S)-configured amidoproline were overlaid with the Pro and Hyp residues within a crystal structure of collagen revealed that the sterically demanding groups point to the outside of these two triple helices and thereby do not interfere with the formation of the triple helix. In all of the other examined collagen derivatives with lower stability of the triple helices, the acetyl or pivaloyl residues point toward the inside of the triple helix and clash with a residue of the neighboring strand. The results also revealed that unfavorable steric dispositions affect the conformational stability of the collagen triple helix more than unfavorable ring puckers of the proline residues. The results are useful for the design of functionalized collagen based materials.

Structural basis for matrix metalloproteinase 1-catalyzed collagenolysis.

The proteolysis of collagen triple-helical structure (collagenolysis) is a poorly understood yet critical physiological process. Presently, matrix metalloproteinase 1 (MMP-1) and collagen triple-helical peptide models have been utilized to characterize the events and calculate the energetics of collagenolysis via NMR spectroscopic analysis of 12 enzyme-substrate complexes. The triple-helix is bound initially by the MMP-1 hemopexin-like (HPX) domain via a four amino acid stretch (analogous to type I collagen residues 782-785). The triple-helix is then presented to the MMP-1 catalytic (CAT) domain in a distinct orientation. The HPX and CAT domains are rotated with respect to one another compared with the X-ray "closed" conformation of MMP-1. Back-rotation of the CAT and HPX domains to the X-ray closed conformation releases one chain out of the triple-helix, and this chain is properly positioned in the CAT domain active site for subsequent hydrolysis. The aforementioned steps provide a detailed, experimentally derived, and energetically favorable collagenolytic mechanism, as well as significant insight into the roles of distinct domains in extracellular protease function.

Preparation and characterization of malonic acid cross-linked chitosan and collagen 3D scaffolds: an approach on non-covalent interactions.

The present study emphasizes the influence of non-covalent interactions on the mechanical and thermal properties of the scaffolds of chitosan/collagen origin. Malonic acid (MA), a bifuncitonal diacid was chosen to offer non-covalent cross-linking. Three dimensional scaffolds was prepared using chitosan at 1.0% (w/v) and MA at 0.2% (w/v), similarly collagen 0.5% (w/v) and MA 0.2% (w/v) and characterized. Results on FT-IR, TGA, DSC, SEM and mechanical properties (tensile strength, stiffness, Young's modulus, etc.) assessment demonstrated the existence of non-covalent interaction between MA and chitosan/collagen, which offered flexibility and high strength to the scaffolds suitable for tissue engineering research. Studies using NIH 3T3 fibroblast cells suggested biocompatibility nature of the scaffolds. Docking simulation study further supports the intermolecular hydrogen bonding interactions between MA and chitosan/collagen.

Role of hydration force in the self-assembly of collagens and amyloid steric zipper filaments.

In protein self-assembly, types of surfaces determine the force between them. Yet the extent to which the surrounding water contributes to this force remains as a fundamental question. Here we study three self-assembling filament systems that respectively have hydrated (collagen), dry nonpolar, and dry polar (amyloid) interfaces. Using molecular dynamics simulations, we calculate and compare local hydration maps and hydration forces. We find that the primary hydration shells are formed all over the surface, regardless of the types of the underlying amino acids. The weakly oscillating hydration force arises from coalescence and depletion of hydration shells as two filaments approach, whereas local water diffusion, orientation, or hydrogen-bonding events have no direct effect. Hydration forces between hydrated, polar, and nonpolar interfaces differ in the amplitude and phase of the oscillation relative to the equilibrium surface separation. Therefore, water-mediated interactions between these protein surfaces, ranging in character from "hydrophobic" to "hydrophilic", have a common molecular origin based on the robustly formed hydration shells, which is likely applicable to a broad range of biomolecular assemblies whose interfacial geometry is similar in length scale to those of the present study.