A hyperstable collagen mimic

BACKGROUND: Collagen is the most abundant protein in animals. Each polypeptide chain of collagen is composed of repeats of the sequence: Gly-X-Y, where X and Y are often L-proline (Pro) and 4(R)-hydroxy-L-proline (Hyp) residues, respectively. These chains are wound into tight triple helices of great stability. The hydroxyl group of Hyp residues contributes much to this conformational stability. The existing paradigm is that this stability arises from interstrand hydrogen bonds mediated by bridging water molecules. This model was tested using chemical synthesis to replace Hyp residues with 4(R)-fluoro-L-proline (Flp) residues. The fluorine atom in Flp residues does not form hydrogen bonds but does elicit strong inductive effects. RESULTS: Replacing the Hyp residues in collagen with Flp residues greatly increases triple-helical stability. The free energy contributed by the fluorine atom in Flp residues is twice that of the hydroxyl group in Hyp residues. The stability of the Flp-containing triple helix far exceeds that of any untemplated collagen mimic of similar size. CONCLUSIONS: Bridging water molecules contribute little to collagen stability. Rather, collagen stability relies on previously unappreciated inductive effects. Collagen mimics containing fluorine or other appropriate electron-withdrawing substituents could be the basis of new biomaterials for restorative therapies.     

Inductive Effects on the Energetics of Prolyl Peptide Bond Isomerization: Implications for Collagen Folding and Stability

The hydroxylation of proline residues in collagen enhances the stability of the collagen triple helix. Previous X-ray diffraction analyses had demonstrated that the presence of an electron-withdrawing substituent on the pyrrolidine ring of proline residues has significant structural consequences [Panasik, N., Jr.; Eberhardt, E. S.; Edison, A. S.; Powell, D. R.; Raines, R. T. Int. J. Pept. Protein Res.1994, 44, 262-269]. Here, NMR and FTIR spectroscopy were used to ascertain kinetic and thermodynamic properties of N-acetyl-[β,γ-(13)C]D,L-proline methylester (1); N-acetyl-4(R)-hydroxy-L-proline [(13)C]methylester (2); and N-acetyl-4(R)-fluoro-L-proline methylester (3). The pK(a)'s of the nitrogen atom in the parent amino acids decrease in the order: proline (10.8) > 4(R)-hydroxy-L-proline (9.68) > 4(R)-fluoro-L-proline (9.23). In water or dioxane, amide I vibrational modes decrease in the order: 1 > 2 > 3. At 37 °C in dioxane, the rate constants for amide bond isomerization are greater for 3 than 1. Each of these results is consistent with the traditional picture of amide resonance coupled with an inductive effect that results in a higher bond order in the amide C=O bond and a lower bond order in the amide C-N bond. Further, at 37 °C in water or dioxane equilibrium concentrations of the trans isomer increase in the order: 1 < 2 < 3. Inductive effects may therefore have a significant impact on the folding and stability of collagen, which has a preponderance of hydroxyproline residues, all with peptide bonds in the trans conformation.     

Effect of 3-hydroxyproline residues on collagen stability

Collagen is an integral part of many types of connective tissue in animals, especially skin, bones, cartilage, and basement membranes. A fibrous protein, collagen has a triple-helical structure, which is comprised of strands with a repeating Xaa-Yaa-Gly sequence. l-Proline (Pro) and 4(R)-hydroxy-l-proline (4-Hyp) residues occur most often in the Xaa and Yaa positions. The 4-Hyp residue is known to increase markedly the conformational stability of a collagen triple helix. In natural collagen, a 3(S)-hydroxy-l-proline (3-Hyp) residue occurs in the sequence: 3-Hyp-4-Hyp-Gly. Its effect on collagen stability is unknown. Here, two host-guest peptides containing 3-Hyp are synthesized: (Pro-4-Hyp-Gly)(3)-3-Hyp-4-Hyp-Gly-(Pro-4-Hyp-Gly)(3) (peptide 1) and (Pro-4-Hyp-Gly)(3)-Pro-3-Hyp-Gly-(Pro-4-Hyp-Gly)(3) (peptide 2). The 3-Hyp residues in these two peptides diminish triple-helical stability in comparison to Pro. This destabilization is small when 3-Hyp is in the natural Xaa position (peptide 1). There, the inductive effect of its 3-hydroxyl group diminishes slightly the strength of the interstrand 3-HypC=O.H-NGly hydrogen bond. The destabilization is large when 3-Hyp is in the nonnatural Yaa position (peptide 2). There, its pyrrolidine ring pucker leads to inappropriate mainchain dihedral angles and interstrand steric clashes. Thus, the natural regioisomeric residues 3-Hyp and 4-Hyp have distinct effects on the conformational stability of the collagen triple helix.     

Ab initio and density functional theory based studies on collagen triplets

An understanding of the amino acid sequence dependent stability of polypeptides is of renowned interest to biophysicists and biochemists, in order to identify the nature of forces that stabilize the three-dimensional structure of proteins. In this study, the role of various collagen triplets influencing the stability of collagen has been addressed. It is found from this study that proline can stabilize the collagen triplet only when other residues are also in the polyproline II conformation. Solvation studies of various triplets indicate that the presence of polar residues increases the free energy of solvation. Especially the triplets containing arginine residues displays a higher solvation free energy. The chemical hardness of all the triplets in collagen-like conformation has been found to be higher than that in the extended conformation. Studies on Gly–X–Y, Gly–X–Hyp, and Gly–Pro–Y triplets confirm that there will be local variations in the stability of collagen along the entire sequence.     

Imino acids and collagen triple helix stability: characterization of collagen-like polypeptides containing Hyp-Hyp-Gly sequence repeats

The analysis of factors contributing to the stability of proteins is a subject of intense debate. Particularly challenging is the study of structural proteins, since their function is their structure. Among these is collagen, the key structural component of bones, skin, cartilage, tendons, and other connecting tissues. It is well established that the collagen triple helix is characterized by the presence of hydroxyproline, whose content modulates triple helix thermal stability according to the requirement of the host organism. Because of the complexity and the fibrous nature of collagen, data on the stability and structure of this protein have been mainly obtained by the use of collagen-like polypeptides. On the basis of CD characterization of collagen-like polypeptides we here show that the presence of Hyp at the X position of repeating triplets Hyp-Hyp-Gly stabilizes the triple helix significantly. This extra-stabilization has been ascribed, by using molecular modeling, to the formation of a hydrogen bond between Hyp residues belonging to the X and the Y positions of adjacent chains. This communication also provides a comprehensive interpretation of the ensemble of available data on polypeptides containing proline derivatives.     

Reciprocity of steric and stereoelectronic effects in the collagen triple helix

In previous work, we demonstrated that 4-fluoroproline residues can contribute greatly to the conformational stability of the collagen triple helix, and that this stability arises from stereoelectronic effects that fix the pucker of the pyrrolidine ring and thereby preorganize the backbone properly for triple-helix formation. Here, we take a reciprocal approach, demonstrating that the steric effect of a 4-methyl group confers stability similar to that from a 4-fluoro group in the opposite configuration. Such fundamental interplay between steric and stereoelectronic effects is heretofore unknown in proteins-natural or synthetic-and provides a new means to modulate conformational stability.     

Rules for the design of aza-glycine stabilized triple-helical collagen peptides

The stability of the triple-helical structure of collagen is modulated by a delicate balance of effects including polypeptide backbone geometry, a buried hydrogen bond network, dispersive interfacial interactions, and subtle stereoelectronic effects. Although the different amino acid propensities for the Xaa and Yaa positions of collagen''s repeating (Glycine–Xaa–Yaa) primary structure have been described, our understanding of the impact of incorporating aza-glycine (azGly) residues adjacent to varied Xaa and Yaa position residues has been limited to specific sequences. Here, we detail the impact of variation in the Xaa position adjacent to an azGly residue and compare these results to our study on the impact of the Yaa position. For the first time, we present a set of design rules for azGly-stabilized triple-helical collagen peptides, accounting for all canonical amino acids in the Xaa and Yaa positions adjacent to an azGly residue, and extend these rules using multiple azGly residues. To gain atomic level insight into these new rules we present two high-resolution crystal structures of collagen triple helices, with the first peptoid-containing collagen peptide structure. In conjunction with biophysical and computational data, we highlight the critical importance of preserving the triple helix geometry and protecting the hydrogen bonding network proximal to the azGly residue from solvent. Our results provide a set of design guidelines for azGly-stabilized triple-helical collagen peptides and fundamental insight into collagen structure and stability.

Guidelines for incorporating aza-glycine residues in collagen peptides are presented, detailing their effects on triple-helical thermal stability.     

Structure and conformation of intramolecularly cross-linked collagen

This work reports the effects of cross-linking agent like formaldehyde (HCHO) and glutaraldehyde (GA) on monomeric collagen and its conformational stability using circular dichroism. The nature of the bonds formed and the stability of the cross-links introduced vary with the aldehyde. The amount of HCHO required is four times greater than GA to induce similar changes in the molar ellipticity. This is due to the structural changes of collagen-aldehyde reaction. The change in the molar ellipticity and Rpn ratio for HCHO and GA treated collagen suggest possible aggregation of collagen molecule. The relative viscosity of native and aldehyde treated collagen was measured and correlated to the changes seen in the CD spectra. The small amount of aldehydes are needed to induce changes in the conformational stability of collagen. This can be applied to the biomaterial and biomedical application.     

A solid-state NMR study of the fast and slow dynamics of collagen fibrils at varying hydration levels

We report solid-state NMR investigations of the effect of temperature and hydration on the molecular mobility of collagen isolated from bovine achilles tendon. (13)C cross-polarization magic angle spinning (MAS) experiments were performed on samples at natural abundance, using NMR methods that detect motionally averaged dipolar interactions and chemical shift anisotropies and also slow reorientational processes. Fast motions with correlation times much shorter than 40 micro s scale dipolar couplings and chemical shift anisotropies of the carbon sites in collagen. These motionally averaged anisotropic interactions provide a measure of the amplitudes of the segmental motions expressed by a molecular order parameter. The data reveal that increasing hydration has a much stronger effect on the amplitude of the molecular processes than increasing temperature. In particular, the Cgamma carbons of the hydroxyproline residues exhibit a strong dependence of the amplitude of motion on the hydration level. This could be correlated with the effect of hydration on the hydrogen bonding structure in collagen, for which this residue is known to play a crucial role. The applicability of 1D MAS exchange experiments to investigate motions on the millisecond time-scale is discussed and first results are presented. Slow motions with correlation times of the order of milliseconds have also been detected for hydrated collagen.     

Increase in scleral collagen stability during glycosylation with threose in vitro

A systematic study of changes in the physicochemical characteristics of scleral collagen in the course of glycosylation by threose, including their dependence on the time changes of transverse cross-linking, was performed. Glycosylation by threose leads to a significant increase in heat, proteolytic, and biomechanical stability of collagen in the scleral tissue and has been shown to be a useful approach for stabilizing scleral collagen. It was found that a fraction of collagen with a reduced denaturation temperature is, apparently, an intermediate in the reaction of glycosylation by threose. The most likely reason for its occurrence is the elongation of the side chains of amino acid residues of the protein in the early stages.     

DOPA Residues Endow Collagen with Radical Scavenging Capacity**

Here we uncover collagen, the main structural protein of all connective tissues, as a redox-active material. We identify dihydroxyphenylalanine (DOPA) residues, post-translational oxidation products of tyrosine residues, to be common in collagen derived from different connective tissues. We observe that these DOPA residues endow collagen with substantial radical scavenging capacity. When reducing radicals, DOPA residues work as redox relay: they convert to the quinone and generate hydrogen peroxide. In this dual function, DOPA outcompetes its amino acid precursors and ascorbic acid. Our results establish DOPA residues as redox-active side chains of collagens, probably protecting connective tissues against radicals formed under mechanical stress and/or inflammation.     

Promoting self-assembly of collagen-related peptides into various higher-order structures by metal-histidine coordination

Collagen is an important and widely used biomaterial and therapeutic. The construction of large-scale collagen structures via the self-assembly of small collagen-related peptides has been extensively studied in the past decade. Here, we report a highly effective and simple means to assemble small synthetic collagen-related peptides into various higher-order structures by utilizing metal-histidine coordination. In this work, two short collagen-related peptides in which histidine residues were incorporated as metal binding sites were designed and chemically synthesized: HG(PPG)(9)GH (X9) and HG(PPG)(4)(PHG)(PPG)(4)GH (PHG). Circular dichroism measurements indicated that these two peptides form only marginally stable collagen triple helices but that their stability can be increased upon the addition of metal ions. Dynamic light scattering analyses, turbidity measurements, TEM, and SEM results demonstrated the metal ion-dependent self-assembly of X9 and PHG into supramolecular structures ranging from various nanofibrils to microscale spherical, laminated, and granulated assemblies. The topology and size of these higher-order structures depends both on the metal ion identity and the location of the binding sites. Most intriguingly, the assembled fibrils show similar D-periodicity to that of natural collagen. Our results demonstrate that metal-histidine coordination can serve as an effective force to induce the self-assembly of unstable collagen-related peptides into higher-order structures.     

Structure-activity studies of 14-helical antimicrobial beta-peptides: probing the relationship between conformational stability and antimicrobial potency

Antimicrobial alpha-helical alpha-peptides are part of the host-defense mechanism of multicellular organisms and could find therapeutic use against bacteria that are resistant to conventional antibiotics. Recent work from Hamuro et al. has shown that oligomers of beta-amino acids ("beta-peptides") that can adopt an amphiphilic helix defined by 14-membered ring hydrogen bonds ("14-helix") are active against Escherichia coli [Hamuro, Y.; Schneider, J. P.; DeGrado, W. F. J. Am. Chem. Soc. 1999, 121, 12200-12201]. We have created two series of cationic 9- and 10-residue amphiphilic beta-peptides to probe the effect of 14-helix stability on antimicrobial and hemolytic activity. 14-Helix stability within these series is modulated by varying the proportions of rigid trans-2-aminocyclohexanecarboxylic acid (ACHC) residues and flexible acyclic residues. We have previously shown that a high proportion of ACHC residues in short beta-peptides encourages 14-helical structure in aqueous solution [Appella, D. H.; Barchi, J. J.; Durell, S. R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 2309-2310]. Circular dichroism of the beta-peptides described here reveals a broad range of 14-helix population in aqueous buffer, but this variation in helical propensity does not lead to significant changes in antibiotic activity against a set of four bacteria. Several of the 9-mers display antibiotic activity comparable to that of a synthetic magainin derivative. Among these 9-mers, hemolytic activity increases slightly with increasing 14-helical propensity, but all of the 9-mers are less hemolytic than the magainin derivative. Previous studies with conventional peptides (alpha-amino acid residues) have provided conflicting evidence on the relationship between helical propensity and antimicrobial activity. This uncertainty has arisen because alpha-helix stability can be varied to only a limited extent among linear alpha-peptides without modifying parameters important for antimicrobial activity (e.g., net charge or hydrophobicity); a much greater range of helical stability is accessible with beta-peptides. For example, it is very rare for a linear alpha-peptide to display significant alpha-helix formation in aqueous solution and manifest antibacterial activity, while the linear beta-peptides described here range from fully unfolded to very highly folded in aqueous solution. This study shows that beta-peptides can be unique tools for analyzing relationships between conformational stability and biological activity.     

Role of length-dependent stability of collagen-like peptides

Understanding the structure, folding, and stability of collagen is complex because of its length and variations in the amino acid (AA) sequence composition. It is well known that the basic constituent of the collagen helix is the triplet repeating sequence of the form Gly-X(AA)-Y(AA). On the basis of previous models and with the frequency of occurrence of the triplets, the ((Gly-Pro-Hyp)n)3 (where n is the number of triplets) sequence replicate has been chosen as the model for the most stable form of the collagen-like sequence. With a view to understand the role of sequence length (or the number of triplets) on the stability of collagen, molecular dynamics simulations have been carried out by varying the number of triplet units on the model collagen-like peptides. The results reveal that five triplets are required to form the stable triple helix. Further analysis shows that the intermolecular structural rigidity of the imino acid residues, hydrogen bonding, and water structure around the three chains of the triple helix play the dominant roles on its structure, folding, and stabilization.     

Collagen stabilization and modification using a polyepoxide, triglycidyl isocyanurate

This paper reports a study on the crosslinking behaviour of dermal collagen with the polyepoxide triglycidyl isocyanurate (TEPIC), which is thought to introduce a specific stabilization effect into a collagen matrix. TEPIC shows a higher reactivity towards collagen compared to glycerol triglycidyl ether, a water-soluble epoxide with the same tri-functionality but having an aliphatic backbone in its molecular structure, and one of the most commonly used epoxide monomers reported in the literature. Significant stabilization of the collagen matrix treated with TEPIC has been shown in our work, based on the examination of hydrothermal properties, thermal degradation and enzyme degradation properties, amino acid analysis and measurement of mechanical properties. Collagen's dimensional stability was also found to be improved, from tensile testing and fibre morphology observations (here by scanning electron microscopy). The overall stabilization effect of TEPIC is comparable or better than the conventionally used crosslinker, glutaraldehyde, and so implies that a stable three-dimensional, covalent network is formed by the TEPIC within the collagen supermolecule, instead of the two-dimensional crosslinked bridging systems in the case of glutaraldehyde. These results also suggest that the additional stabilization by TEPIC may relate to its triazine nucleus, which introduces a more rigid conformation into the collagen polypeptide chains. In addition, the treated collagen matrix has been found to have increased adsorption ability for anionic dyes. This study has shown that TEPIC can be considered as one of the more effective aldehyde alternatives, which could have a potential significance in leather and textile industrial applications.     

Peptides that anneal to natural collagen in vitro and ex vivo

Collagen comprises ? of the protein in humans and ? of the dry weight of human skin. Here, we implement recent discoveries about the structure and stability of the collagen triple helix to design new chemical modalities that anchor to natural collagen. The key components are collagen mimetic peptides (CMPs) that are incapable of self-assembly into homotrimeric triple helices, but are able to anneal spontaneously to natural collagen. We show that such CMPs containing 4-fluoroproline residues, in particular, bind tightly to mammalian collagen in vitro and to a mouse wound ex vivo. These synthetic peptides, coupled to dyes or growth factors, could herald a new era in assessing or treating wounds.     

How stable is a collagen triple helix? An ab initio study on various collagen and β‐sheet forming sequences

Collagen forms the well characterized triple helical secondary structure, stabilized by interchain H-bonds. Here we have investigated the stability of fully optimized collagen triple helices and beta-pleated sheets by using first principles (ab initio and DFT) calculations so as to determine the secondary structure preference depending on the amino acid composition. Models composed of a total of 18 amino acid residues were studied at six different amino acid compositions: (i) L-alanine only, (ii) glycine only, (iii) L-alanines and glycine, (iv) L-alanines and D-alanine, (v) L-prolines with glycine, (vi) L-proline, L-hydroxyproline, and glycine. The last two, v and vi, were designed to mimic the core part of collagen. Furthermore, ii, iii, and iv model the binding and/or recognition sites of collagen. Finally, i models the G-->A replacement, rare in collagen. All calculated structures show great resemblance to those determined by X-ray crystallography. Calculated triple helix formation affinities correlate well with experimentally determined stabilities derived from melting point (T(m)) data of different collagen models. The stabilization energy of a collagen triple helical structure over that of a beta-pleated sheet is 2.1 kcal mol(-1) per triplet for the [(-Pro-Hyp-Gly-)(2)](3) collagen peptide. This changes to 4.8 kcal mol(-1) per triplet of destabilization energy for the [(-Ala-Ala-Gly-)(2)](3) sequence, known to be disfavored in collagen. The present study proves that by using first principles methods for calculating stabilities of supramolecular complexes, such as collagen and beta-pleated sheets, one can obtain stability data in full agreement with experimental observations, which envisage the applicability of QM in molecular design.     

Fluorescence characterization of the thermal stability of collagen mimic peptides

The thermal stability of triple helical structure plays a critical role in collagen biosynthesis, function and degradation. CD technique was utilized to characterize the thermal stability of synthetic collagen mimic peptides. Fluorescence spectroscopy is widely used with easy access all around the world because of its inexpensive instrumentation, low operation cost, easy operation, and high sensitivity. Here we have developed an alternative fluorescence method to detect the thermal stability of collagen mimic peptides. We have demonstrated that fluorescence spectroscopy could measure the thermal stability of collagen mimic peptides with low concentrations under different circumstances. This highly sensitive fluorescence self-quenching assay will greatly expedite the studies of sequence-dependent properties of collagen mimic peptides, and it has great potential in the application of determining the thermal stability of triple helix systems such as collagens, collectins, adiponectin, macrophage scavenger and C1q.     

Effect of UV irradiation on stabilized collagen: role of chromium(III)

Research on the effect of UV radiation on stabilized collagen is an area of potential interest owing to the fact that collagen is an important biomaterial finding immense use in various fields. In this present study, effect of UV irradiation on collagen stabilized using chromium(III) has been studied. The physical and optical properties affected by UV irradiation have been detailed. Viscosity measurements have shown that chromium(III) treated collagen has better stability against UV radiation than native collagen. Circular dichroic studies indicate that increase in concentration of chromium(III) does not affect the conformation of collagen however, the duration of irradiation has profound impact on the conformation of collagen. The fluorescence intensity of native collagen has been found to decrease more than that of chromium(III) treated collagen. The difference absorption spectra also shows that chromium(III) treatment brings about more stability to collagen against UV irradiation.     

Triple-helix propensity of hydroxyproline and fluoroproline: comparison of host-guest and repeating tripeptide collagen models

Peptide models have proved important in defining the structural features of the collagen triple-helix. Some models are based on multiple repeats of a given tripeptide unit, while a host-guest design includes an individual tripeptide unit substituted within a constant repeating Pro-Hyp-Gly framework. In the present study, proline, hydroxyproline, and fluoroproline residues are incorporated in X- or Y-positions of a guest triplet in the host-guest peptide design. All host-guest peptides, including Hyp-Pro-Gly, formed stable triple-helices, even though a triple-helix cannot be formed by (Hyp-Pro-Gly)10. The order of stability Pro-Hyp-Gly > Pro-Pro-Gly > Hyp-Pro-Gly remains the same in all models, while the Pro-Flp-Gly is very stabilizing in a repeating context but destabilizing in a host-guest context. The range of thermal stabilities and calorimetric enthalpies is very small among the five host-guest peptides, consistent with the concept that the effect of one Xaa-Yaa-Gly tripeptide unit in the host-guest system would be less than the much larger variations when there are 10 repeating units. However, a simple additive model based on host-guest peptides predicts a greater stability than experimentally observed. The difference in stability contributions of the same tripeptide unit in host-guest versus repeating tripeptide systems illustrates the impact of sequence environment on stability, and factors that play a role include ring puckering as a consequence of electron inductive effects, residual monomer structure, and native state hydration networks.