Solid-Phase synthesis and stability of triple-helical peptides incorporating native collagen sequences

A generally applicable solid-phase methodology has been developed for the synthesis of triple-helical polypeptides incorporating native collagen sequences. Three nascent peptide chains are C-terminal linked through one N^α-amino and two N^ε-amino groups of Lys, while repeating Gly-Pro-Hyp triplets induce triple helicity. Different protecting group strategies, including several three-dimensionally orthogonal schemes, have been utilized for the synthesis of four homotrimeric triple-helical polypeptides (THPs) of 79–124 residues, three of which incorporate native type IV collagen sequences. Highly efficient assemblies were achieved by 9-fluorenylmethoxycarbonyl (Fmoc) N^α-amino group protection, in situ 2-(1H-benzotriazole-l-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate mediated couplings, and 1,8-diazabicyclo [5.4.0] undec-7-ene mediated Fmoc group removal. THPs were characterized by Edman degradation sequencing, size-exclusion chromatography, mass spectrometry, reversed-phase high performance liquid chromatography, and CD spectroscopy. THP thermal stabilities ranged from 35 to 59°C, with chain length and Hyp content being the influential factors. Melting temperatures and van't Hoff enthalpies for peptide triple-helical denaturation could be correlated well to Hyp content. The THP synthetic protocol developed here will allow for the study of both structure and biological activity of specific collagen sequences in homotrimeric and heterotrimeric forms. © 1993 John Wiley & Sons, Inc.     

Helical twists of collagen model peptides

Average helical twists were calculated by the method of Sugeta and Miyazawa (Biopolymers 1967, 5, 673-679) for all of the collagen model peptides analyzed to date. Calculation of the helical twists of all triplets in each peptide strand provided novel insights for several model peptides. In the (Pro-Pro-Gly)n (n = 9 and 10), the helical twists showed cyclic fluctuations between 40 and 65 degrees with a 20 A period, suggesting that their molecular conformations were close enough to the ideal 7/2-helix to show the helical repeat of 20 A. Rather small helical twists in the guest regions of IBP in complex and T3-785 were attributed to the interaction with Integrin I domain and a relaxed conformation caused by three consecutive triplets lacking imino acid residues, respectively. Although most of the triplets used in this study were imino acid-rich triplets, helical twists were scattered in a wide range from 30 to 70 degrees with an overall average of 52.6 degrees . This distribution of helical twists indicated a strong preference for the 7/2-helical conformation (51.4 degrees ) rather than the 10/3-helical model (36 degrees ).     

UV damage of collagen: insights from model collagen peptides

Fibrils of Type I collagen in the skin are exposed to ultraviolet (UV) light and there have been claims that collagen photo-degradation leads to wrinkles and may contribute to skin cancers. To understand the effects of UV radiation on collagen, Type I collagen solutions were exposed to the UV-C wavelength of 254 nm for defined lengths of time at 4°C. Circular dichroism (CD) experiments show that irradiation of collagen leads to high loss of triple helical content with a new lower thermal stability peak and SDS-gel electrophoresis indicates breakdown of collagen chains. To better define the effects of UV radiation on the collagen triple-helix, the studies were extended to peptides which model the collagen sequence and conformation. CD studies showed irradiation for days led to lower magnitudes of the triple-helix maximum at 225 nm and lower thermal stabilities for two peptides containing multiple Gly-Pro-Hyp triplets. In contrast, the highest radiation exposure led to little change in the T(m) values of (Gly-Pro-Pro)(10) and (Ala-Hyp-Gly)(10) , although (Gly-Pro-Pro)(10) did show a significant decrease in triple helix intensity. Mass spectroscopy indicated preferential cleavage sites within the peptides, and identification of some of the most susceptible sites of cleavage. The effect of radiation on these well defined peptides gives insight into the sequence and conformational specificity of photo-degradation of collagen.     

Equilibrium thermal transitions of collagen model peptides

The folding of collagen in vitro is very slow and presents difficulties in reaching equilibrium, a feature that may have implications for in vivo collagen function. Peptides serve as good model systems for examining equilibrium thermal transitions in the collagen triple helix. Investigations were carried out to ascertain whether a range of synthetic triple-helical peptides of varying sequences can reach equilibrium, and whether the triple helix to unfolded monomer transition approximates a two-state model. The thermal transitions for all peptides studied are fully reversible given sufficient time. Isothermal experiments were carried out to obtain relaxation times at different temperatures. The slowest relaxation times, on the order of 10-15 h, were observed at the beginning of transitions, and were shown to result from self-association limited by the low concentration of free monomers, rather than cis-trans isomerization. Although the fit of the CD equilibrium transition curves and the concentration dependence of T(m) values support a two-state model, the more rigorous comparison of the calorimetric enthalpy to the van't Hoff enthalpy indicates the two-state approximation is not ideal. Previous reports of melting curves of triple-helical host-guest peptides are shown to be a two-state kinetic transition, rather than an equilibrium transition.     

Conformational change of the triple-helical structure. I. Synthesis of model peptides of collagen by the solid-phase method

As model peptides of collagen, (Pro-Pro-Gly)_n (n = 10, 12, 14, and 15) and (Pro-Pro-Gly)_n(Ala-Pro-Gly)_m(Pro-Pro-Gly)_n (2n + m = 15; m = 1, 3, and 5) were synthesized by the solid-phase method. The final products were pure when checked by high-voltage paper electrophoresis and by amino acid analysis. Elemental composition was also examined.     

Cleavage of native type III collagen in the collagenase susceptible region by thermolysin.

Viscometric assays were used to demonstrate the activity of thermolysin (EC 3.4.24.4) on native type III collagen in solution. Analysis of the reaction products by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electron microscopic visualisation of segment long spacing aggregates demonstrated localised cleavage of the collagen in the collagenase susceptible region.     

Activity of matrix metalloproteinase-9 against native collagen types I and III

Interstitial collagen types I, II and III are highly resistant to proteolytic attack, due to their triple helical structure, but can be cleaved by matrix metalloproteinase (MMP) collagenases at a specific site, approximately three-quarters of the length from the N-terminus of each chain. MMP-2 and -9 are closely related at the structural level, but MMP-2, and not MMP-9, has been previously described as a collagenase. This report investigates the ability of purified recombinant human MMP-9 produced in insect cells to degrade native collagen types I and III. Purified MMP-9 was able to cleave the soluble, monomeric forms of native collagen types I and III at 37 degrees C and 25 degrees C, respectively. Activity against collagens I and III was abolished by metalloproteinase inhibitors and was not present in the concentrated crude medium of mock-transfected cells, demonstrating that it was MMP-9-derived. Mutated, collagenase-resistant type I collagen was not digested by MMP-9, indicating that the three-quarters/one-quarter locus was the site of initial attack. Digestion of type III collagen generated a three-quarter fragment, as shown by comparison with MMP-1-mediated cleavage. These data demonstrate that MMP-9, like MMP-2, is able to cleave collagens I and III in their native form and in a manner that is characteristic of the unique collagenolytic activity of MMP collagenases.     

The chain register in heterotrimeric collagen peptides affects triple helix stability and folding kinetics

Collagen type IV is a highly specialized form of collagen found only in basement membranes, where it provides mechanical stability and structural integrity to tissues and organs, and binding sites for cell adhesion. In its ubiquitous form, collagen type IV consists of two alpha1 chains and one alpha2 chain, whose internal alignment within the triple helix seems to exert a strong influence on the binding affinity to alpha1beta1 integrin receptor. This has been assessed recently using two synthetic collagen peptides that contain the cell adhesion epitope of collagen type IV and are assembled into the most plausible alpha1alpha2alpha1' and alpha2alpha1alpha1' registers. In the present study, the effects of the chain register on the stability of the triple helix and the folding kinetics of these collagen peptides were investigated by CD spectroscopy and microcalorimetry. The results revealed a multi-domain structural organization for both trimers, with an unexpected strong effect of the chain alignment on the conformational stability. Molecular dynamics simulations served to rationalize more properly the experimental results.     

Sequence-specific liquid crystallinity of collagen model peptides. I. Transmission electron microscopy studies of interfacial collagen gels

The conformation, crystal structure and self-assembly behavior of three peptides with collagen-like repetitive sequences [(1) peptide GAPGPP: (Glu)(5)(Gly-Ala-Pro-Gly-Pro-Pro)(6)(Glu)(5); (2) peptide GVPGPP: (Glu)(5)(Gly-Val-Pro-Gly-Pro-Pro)(6)(Glu)(5); and (3) peptide GAPGPA: (Glu)(5)(Gly-Ala-Pro-Gly-Pro-Ala)(6)(Glu)(5)] were compared. The peptides were characterized using transmission electron microscopy, electron diffraction, environmental scanning electron microscopy, and Fourier transform ir spectroscopy in order to determine how the molecular geometry dictated by each sequence affects the spontaneous generation of long-range ordered structures. Samples of each peptide, at ambient temperature and at 5 degrees C, were examined as films dried from aqueous solution, air-water interfacial films, and chloroform-water interfacial films. Peptide GAPGPP prepared at 5 degrees C and dried from bulk solution was found to have a collagen-like triple-helical structure. A sinusoidally textured gel, suggestive of cholesteric behavior was observed for peptides GAPGPP and GVPGPP at the aqueous chloroform interface at 5 degrees C. Peptide GAPGPA also formed a gel, but less reproducibly and the sinusoidal texture was not as well defined. The periodicities of the sinusoidal textures were reproducibly 10 microm for peptide GAPGPP, 7 microm for peptide GVPGPP, and 6 microm for peptide GAPGPA. The differences in the periodicity of the banded structure and in the crystallization behavior of the three peptides is attributed to differences in the symmetry of the preferred packing arrangement for each peptide, as evidenced by electron diffraction from crystallites that coexist with the sinusoidal gel. These differences are believed to be a measure of the effective symmetry and shape of the molecular cross section.     

Identification of cyanogen bromide peptides involved in intermolecular cross-linking of bovine type III collagen.

Cyanogn bromide peptides derived from bovine type III collagen and containing reducible cross-links were isolated and identified. Two peptides, alpha 1 (III)CB7 and alpha 1 (III)CB9B, from within the helical portion of the molecule were shown to contain the 'amino donor' residues cross-linked to non-helical 'aldehyde donor' residues in the formation of cross-links. This information, in conjunction with previously published data for the order of the cyanogen bromide peptides [Fietzek, Allman, Rauterberg & Wachter (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 84-86], suggests that in type III collagen intermolecular cross-links are located in the end-overlap regions, so as to stabilize a quarter-stagger arrangement of molecules within the fibre in a similar manner to that proposed for type I and type II collagens.     

Conformation of histidine model peptides. III. Chiroptical properties of cyclo-L-alanyl-L-histidine and cyclo-L-histidinyl-L-histidine

The chiroptical properties of the cyclic dipeptides cyclo-L -alanyl-L -histidine and cyclo-L -histidinyl-L -histidine have been investigated as a function of molecular conformation. The rotatory strengths of the n-π* transitions of the peptide chromophores and the lowest energy π-π* transitions of the imidazole chromophores have been calculated as a function of the angle of fold of the cyclic dipeptide group and the dihedral angles χ¹ and χ² of the amino acid side chains. The results of this investigation are consistent with the preferred position of the dihedral angle χ¹ occurring near 60° in the free base form of cyclo-L -alanyl-L -histidine, and near 180° when the imidazole side chain is protonated. Furthermore, in the case of the free base form of the imidazole group, it is possible that the tautomeric isomer in which N^ε is protonated may be more prevalent than the isomer in which N^δ is protonated.     

Heterotrimeric collagen peptides containing functional epitopes. Synthesis of single‐stranded collagen type I peptides related to the collagenase cleavage site

Synthetic collagen peptides containing larger numbers of Gly‐Pro‐Hyp repeats are difficult to purify by standard chromatographic procedures. Therefore, efficient strategies are required for the synthesis of higher molecular weight collagen‐type peptides. Applying the Fmoc/tBu chemistry, a comparative analysis of the standard stepwise chain elongation procedure on solid support with the procedure based on the use of the synthons Fmoc‐Gly‐Pro‐Hyp(tBu)‐OH and Fmoc‐Pro‐Hyp‐Gly‐OH was performed. The crude products resulting from the stepwise elongation procedure and from the use of Fmoc‐Gly‐Pro‐Hyp(tBu)‐OH clearly revealed large amounts of microheterogeneities that result from incomplete imino acid acylation as well as from diketopiperazine formation with cleavage of Gly‐Pro units from the growing peptide chain. Conversely, by the use of the Fmoc‐Pro‐Hyp‐Gly‐OH synthon, the quality of the crude products was significantly improved; moreover, protection of the Hyp side chain hydroxyl function is not required using the Fmoc/tBu strategy. With this optimized synthetic procedure, relatively large collagen‐type peptides were obtained in satisfactory yields as highly homogeneous compounds. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of peptides by the solid-phase method. III. Bradykinin: fragments and analogs

The natural sequence of bradykinin (BK) and 55 fragments or analogs of this peptide were perpared via the solid-phase method. The peptides were purified using ion-exchange (O-carboxymethyl(CM) and partition (Sephadex G-25) chromatography. The purity of each peptide was established by paper and thin-layer chromatography, paper electrophoresis, amino acid analysis, and biological assays. The compounds were tested in anesthetized rats (tested in vivo) and in two smooth-muscle preparations (rabbit aorta strip, cat ileum strip) in which BK produces contraction by stimulating specific receptors of different types. Some of the new peptides are interesting in that they either resist pulmonary inactivation, or are more potent than BK itself, or antagonize the myotropic effect of BK in rabbit aorta strips.     

Molecular basis of co-operativity in protein folding. III. Structural identification of cooperative folding units and folding intermediates.

The hierarchical partition function formalism for protein folding developed earlier has been extended through the use of three-dimensional polar and apolar contact plots. For each amino acid residue in the protein, these plots indicate the apolar and polar surfaces that are buried from the solvent, the identity of all amino acid residues that contribute to this shielding, and the magnitude of their contributions. These contact plots are then used to examine the distribution of the free energy of stabilization throughout the protein molecule. Analysis of these data allows identification of co-operative folding units and their hierarchical levels, and the identification of partially folded intermediates with a significant probability of being populated. The overall folding/unfolding thermodynamics of 12 globular proteins, for which crystallographic and experimental thermodynamics are available, is predicted within error. An energetic classification of partially folded intermediates is presented and the results compared to those cases for which structural and thermodynamic experimental information is available. Four different types of partially folded states and their structural energies are considered. (1) Local intermediates, in which only a local region of the protein loses secondary and tertiary interactions, while the rest of the protein remains intact. (2) Global intermediates, corresponding to the standard molten globule definition, in which significant secondary structure is maintained but native-like tertiary structure contacts are disrupted. (3) Extended intermediates characterized by the existence of secondary structure elements (e.g. alpha-helices) exposed to solvent. (4) Folding intermediates in proteins with two structural domains. The structure and energetics of folding intermediates of apo-myoglobin, alpha-lactalbumin, phosphoglycerate kinase and arabinose-binding protein are considered in detail.     

Purification and characterization of native type XIV collagen.

A new molecule, type XIV collagen, with domains homologous to type IX and XII collagens has been recently discovered in pepsin extracts of fetal bovine tissues (Dublet, B., and van der Rest, M. (1991) J. Biol. Chem. 266, 6853-6858). In the present study, we describe the purification and the characterization of the intact native form of this newly discovered collagen. By using only two chromatographic steps we were able to obtain pure type XIV collagen. Furthermore, minor modifications of the protocol allowed us to perform the simultaneous large scale purification of type XII and type XIV collagens from the same tissue. Intact type XIV collagen migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as two bands of 220 and 290 kDa (reducing conditions). After collagenase treatment, a single band of 190 kDa is observed, which represents the large non-collagenous domain of the molecule (NC3). Rotary shadowing electron micrographs of intact type XIV collagen show a cross-shaped structure formed by a thin tail attached through a central globule to three identical "fingers." These properties are similar to those previously described for intact chicken type XII collagen (Dublet, B., Oh, S., Sugrue, S. P., Gordon, M. K., Gerecke, D. R., Olsen, B. R., and van der Rest, M. (1989) J. Biol. Chem. 264, 13150-13156), but the two molecules are different gene products and have charge and glycosylation differences. Finally, we show that the three chains of purified type XIV collagen have an apparent molecular mass of approximately 220 kDa and are not cross-linked to each other by bonds other than disulfide bridges. The same observation was made for type XII collagen. In both cases, the 290-kDa migrating band in SDS-PAGE is due to incomplete denaturation in electrophoresis sample buffer in the absence of urea.     

Synthesis of heterotrimeric collagen peptides containing the alpha1beta1 integrin recognition site of collagen type IV.

Collagen type IV provides a biomechanically stable scaffold into which the other constituents of basement membranes are incorporated, but it also plays an important role in cell adhesion. This occurs with collagen type IV mainly via the alpha1beta1 integrin, and the proposed epitope involved in this type of collagen/integrin interaction corresponds to a non-sequential R/Xaa/D motif, where the arginine and aspartate residues are provided by the alpha2 and alpha1 chains of the collagen molecule, respectively. Since the stagger of the three alpha chains in native collagen type IV is still unknown and different alignments of the chains lead to different spatial epitopes, two heterotrimeric collagen peptides containing the natural 457-469 sequences of the cell adhesion site were synthesized in which the single chains were assembled via disulfide bonds into the two most plausible alpha1alpha2alpha1' and alpha2alpha1alpha1' registers. The differentiated triple-helical stabilities of the two heterotrimers suggest a significant structural role of the chain register in collagen, although the binding to alpha1beta1 integrin is apparently less affected as indicated by preliminary experiments.     

Lysine proximity significantly affects glycation of lysine-containing collagen model peptides

Advanced glycation end products (AGE) are known to cause diabetes complications in hyperglycemia patients. In this study we prepared hetero-trimers of collagen model peptides comprising Ac-(Pro-Hyp-Gly)(5)-Pro-Lys-Gly-(Pro-Hyp-Gly)(5)-Ala-NH(2) (4) and Ac-(Pro-Hyp-Gly)(11)-Ala-NH(2) (5) to investigate the clustering effect of lysine on AGE formation. The formation rate of carboxymethyllysine over several months was determined for the mixtures of peptides 4 and 5 at (3:0), (2:1) and (1:2) in the presence of glucose. The contents of carboxymethyllysine were significantly enhanced for (3:0) and (2:1) as compared with (1:2), suggesting that the proximity of lysine residues in the trimers accelerated formation of the AGE. Furthermore, a lysine dimerization moiety (GOLD) was identified for the first time from AGEs of glucose origin, which implied the significance of GOLD in oligomerization of collagens and other long-life proteins.