Polymeric C-terminal cross-linked material from type-I collagen. A modified method for purification, anomalous behaviour on gel filtration, molecular weight estimation, carbohydrate content and lipid content.

Polymeric cross-linked C-terminal peptide material (poly-alpha 1CB6) from mature bovine tendon type-I collagen was prepared and purified by a modification of the method previously described [Light & Bailey (1980) Biochem. J. 185, 373-381]. Poly-alpha 1CB6 was shown to exhibit concentration-dependent aggregation effects on gel filtration due to interaction with a filtration medium. The material had an amino acid content that was very similar to a mixture of alpha 1CB6 and alpha 1CB5. The material was shown to be polydisperse with a mol.wt. range of 50 000-350 000, but chromatographic fractions were relatively homogeneous over this molecular weight range with respect to amino-acid composition. The heterogeneity of the material was not due to incomplete CNBr peptide cleavage, as poly-alpha 1CB6 did not contain detectable quantities of methionine. The material showed no discrete bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but gave a constant blue stain throughout the molecular weight range described above. Lipid analysis showed that the partially purified material contained elevated levels of stearate when compared to the crude CNBr-digested starting material. This may indicate the specific association of a stearic-acid-rich lipid with the peptide material. On carbohydrate analysis poly-alpha 1CB6 was shown to contain only galactose and glucose at levels of 0.72 and 0.28% respectively. The carbohydrate and amino acid analyses indicated that (alpha 1CB6)2-(alpha 1CB5)1 may be the basic cross-linked structural unit of poly-alpha 1CB6)2-(alpha 1CB5)1 units, although the carbohydrate analysis indicated that the higher molecular weight oligomers may be enriched in alpha 1CB6.     

Covalent structure of collagen. Amino acid sequence of an arthritogenic cyanogen bromide peptide from type II collagen of bovine cartilage

Bovine articular type II collagen was prepared by limited pepsin digestion, differential salt fractionation and carboxymethylcellulose chromatography. Cyanogen bromide digestion of purified type II collagen alpha chains yielded twelve distinct peptides designated CB1-12. The peptide alpha 1(II)-CB11 was isolated by carboxymethylcellulose chromatography and Sephadex G-75S gel filtration. Automated Edman degradation together with chymotrypsin, thermolysin and trypsin digestion enabled identification of its complete amino acid sequence. Compared with type I and type III collagen, the data show similarity with alpha 1(I)-CB8 and alpha 1(III)-CB6-1-8-10-2 peptides, respectively. The peptide is located within residues 124-402 of the alpha 1(II) collagen chain and with its identification, now extends the known amino acid sequence of bovine type II cartilage collagen to 660 amino acid residues including alpha 1(II)-CB1-2-6-12-11-8-10 (partial). This corresponds to alpha 1(I)-CB0-1-2-4-5-8-3-7 (partial; 1-660) and alpha 1(III)-CB3A-3B-3C-7-6-1-8-10-2-4-5 (partial; 1-660) of bovine alpha 1(I) and alpha 1(III) collagen chains.     

Nonenzymatic glycation of type I collagen. The effects of aging on preferential glycation sites.

The present study was designed to investigate the effects of aging on preferential sites of glucose adduct formation on type I collagen chains. Two CNBr peptides, one from each type of chain in the type I tropocollagen molecule, were investigated in detail: alpha 1(I)CB3 and alpha 2CB3-5. Together these peptides comprise approximately 25% of the total tropocollagen molecule. The CNBr peptides were purified from rat tail tendon, obtained from animals aged 6, 18, and 36 months, by ion exchange chromatography, gel filtration, and high-performance liquid chromatography (HPLC). Sugar adducts were radiolabeled by reduction with NaB3H4. Glycated tryptic peptides were prepared from tryptic digests of alpha 2CB3-5 and alpha 1(I)CB3 by boronate affinity chromatography and HPLC. Peptides were identified by sequencing and by compositional analysis. Preferential sites of glycation were observed in both CB3 and alpha 2CB3-5. Of the 5 lysine residues in CB3, Lys-434 was the favored glycation site. Of the 18 lysine residues and 1 hydroxylysine residue in alpha 2CB3-5, 3 residues (Lys-453, Lys-479, and Lys-924) contained more than 80% of the glucose adducts on the peptide. Preferential glycation sites were highly conserved with aging. In collagen that had been glycated in vitro, the relative distribution of glucose adducts in old animals differed from that of young animals. In vitro experiments suggest that primary structure is the major determinant of preferential glycation sites but that higher order structure may influence the relative distribution of glucose adducts among these preferred sites.     

Intermolecular cross-linking and stereospecific molecular packing in type I collagen fibrils of the periodontal ligament

A trypsin digest of denatured NaB3H4-reduced native bovine periodontal ligament was prepared and fractionated by gel filtration and cellulose ion-exchange column chromatography. Prior to trypsin digestion, a complete acid hydrolysate was subjected to analyses for nonreducible stable and reducible intermolecular cross-links. Minute amounts of the former and significant amounts of the reduced cross-links dihydroxylysinonorleucine (1.1 mol/mol of collagen), hydroxylysinonorleucine (0.9 mol/mol of collagen), and histidinohydroxymerodesmosine (0.6 mol/mol of collagen) were found. The covalent intermolecular cross-linked two-chained peptides that were isolated were subjected to amino acid and sequence analyses. The structures for the different two-chained linked peptides were alpha 1CB4-5(76-90)[Hyl-87] X alpha 1CB6-(993-22c)[Lysald-16c], alpha 1CB4-5(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Hylald-16c], alpha 2CB4(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Lysald-16c], and alpha 2CB4(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Hylald-16c]. The cross-link in each peptide was glycosylated. This is the first characterization by sequence analysis of a cross-link involving Hyl-87 in an alpha 2 chain in collagen. A stoichiometric conversion of residue 16c aldehyde to an intermolecular cross-link in each of the COOH-terminal nonhelical peptide regions of both alpha 1 chains in a molecule of type I collagen was found. The ratio of alpha 1 to alpha 2 intermolecularly cross-linked chains involved was 3.3:1, indicating a stereospecific three-dimensional molecular packing of type I collagen molecules in bovine periodontal ligament.     

Cross-linking of collagen. Location of pyridinoline in bovine articular cartilage at two sites of the molecule.

The location of pyridinoline in 18-month-old bovine articular cartilage was investigated by fractionation of CNBr-derived peptides by ion-exchange chromatography and gel filtration. Two peptides, PCP1 and PCP2, were isolated and were shown to contain stoichiometric amounts of pyridinoline. From its amino acid composition and sequence studies, peptide PCP1 was shown to comprise two C-terminal non-helical chains (CB14) linked through pyridinoline to the alpha 1(II)-CB12 portion of the helix. The CB14 chains appeared to be labile at their C-terminal ends, resulting in lower-than-expected amounts of homoserine, and only the N-terminal portion of the peptide was sequenced. Similar studies of peptide PCP2 showed that it contained two N-terminal non-helical chains (CB4) linked to the alpha 1(II)-CB9,7 portion of the helix. The isolated peptides therefore confirmed the function of pyridinoline in stabilizing the 4D stagger of adjacent molecules. The possibility that the cross-link could act both as an intra- and an inter-microfibrillar cross-link was considered. A mechanism of formation of pyridinoline was postulated that, together with other evidence, appears to support the view that, in cartilage, pyridinoline acts primarily as an intramicrofibrillar cross-link and does not contribute to increased stability during maturation through lateral aggregation and bonding of filaments.     

Collagen cross-linking. Isolation of cross-linked peptides from collagen of chicken bone

Cross-linked peptides were isolated from chicken bone collagen that had been digested with CNBr or with bacterial collagenase. Analyses of (3)H radioactivity in disc electrophoretic profiles of the CNBr peptides from bone collagens that had been treated with NaB(3)H indicated that a major site of intermolecular cross-linking in chicken bone collagen is located between the carboxy-terminal region of an alpha1 chain and a small CNBr peptide, probably situated near the amino-terminus of an alpha1 or alpha2 chain in an adjacent collagen molecule. A small amount of this cross-linked CNBr peptide was isolated from a CNBr digest of chicken bone collagen by column chromatography. Amino acid analysis showed that the CNBr peptide, alpha1CB6B, the carboxy-terminal peptide of the alpha1 chain, was the major CNBr peptide in the preparation, and the reduced cross-linking components were identified as hydroxylysinohydroxynorleucine (HylOHNle), with a smaller amount of hydroxylysinonorleucine (HylNle). However, the composition and the low recovery of the cross-linking amino acids suggested that the preparation was a mixture of CNBr peptides alpha1CB6B and alpha1CB6B cross-linked to a small CNBr peptide whose identity could not be determined. A small cross-linked peptide was isolated from chicken bone collagen that had been reduced with NaB(3)H(4) and digested with bacterial collagenase. This peptide was the major cross-linked peptide in the digest and contained a stoicheiometric amount of the reduced cross-linking compounds. A peptide which had the same amino acid composition, but contained the cross-linking compounds in their reducible forms, was isolated from a collagenase digest of chicken bone collagen that had not been treated with NaBH(4). The absence of the reduced cross-links from this peptide indicates that, at least for the cross-linking site from which the peptide derives, natural reduction is not a significant pathway for biosynthesis of stable cross-links. However, most of the reducible cross-linking component in the peptide appeared to stabilize in the bone collagen by rearrangement from aldimine to ketoamine form.     

Ehlers-Danlos syndrome type VIIB. Deletion of 18 amino acids comprising the N-telopeptide region of a pro-alpha 2(I) chain

A patient with Ehlers-Danlos syndrome Type VIIB was found to have an interstitial deletion of 18 amino acids in approximately half of the pro-alpha 2(I) chains of Type I procollagen. Analysis of pepsin-solubilized tissue and fibroblast collagen revealed an abnormal additional chain, alpha 2(I)', which migrated in sodium dodecyl sulfate-5% polyacrylamide gel electrophoresis between the normal alpha 1(I) and alpha 2(I) chains. The apparent ratio of normal alpha 1(I):mutant alpha 2(I)':normal alpha 2(I) was 4:1:1. Procollagen studies and enzyme digestion studies of native mutant collagen suggested defective removal of the amino propeptide. Sieve chromatography of CNBr peptides from purified alpha 2(I)' chains revealed the absence of the normal amino telopeptide fragment CB 1 and the appearance of a larger new peptide of approximately 60 residues (CB X). Compositional and sequencing studies of this peptide identified normal amino propeptide sequences. However, the most carboxyl-terminal tryptic peptide of CB X differed substantially in composition and sequence from the expected and was found to have an interstitial deletion of 18 amino acids corresponding to the N-telopeptide of the pro-alpha 2(I) chain. This deletion removes the normal sites of cleavage of the N-proteinase and also removes a critical cross-linking lysine residue. The 18 amino acids deleted correspond exactly to the residues encoded by exon 6 of the pro-alpha 2(I) collagen gene (COL 1 A2), and, therefore, the protein defect may be due to a genomic deletion, or alternatively, an RNA splicing defect.     

The identity of a cyanogen bromide fragment of bovine dentin collagen containing the site of an intermolecular cross-link.

A peptide fraction isolated from a cyanogen bromide digest of bovine dentin collagen had a molecular weight of 46000. Its size and amino acid composition indicated that it could not consist of peptides derived from the cleavage of a single alpha chain. On reduction with tritiated sodium borohydride, radioactivity was incorporated primarily into 5, 5'-dihydroxylysinonorleucine without degradation at the peptide backbone. Periodate cleavage of the reduced or nonreduced peptide fraction generated one fragment of molecular weight 28000 and one of 18000 completely accounting for the size of the parent peptide. On amino acid analysis the constituent single-chain peptides were determined to be alpha2CB4 and alpha1CB6. Both peptides isolated after periodate oxidation of the tritiated borohydride reduced cross-link peptide were found to contain (3H)hydroxynorvaline. These data show that some hydroxylysine of alpha2CB4, a helical region peptide, was present in aldehyde form and could act as the aldehyde donor icross-link, Schiff's base formation. The only cross-linkage of this alpha2CB4 acting as an aldehyde donor peptide to alpha1CB6 would be a helical region to helical region bond, perhaps accounting for the unusual stability and low solubility of dentin collagen.     

Type II collagen arthritis: identification of arthritogenic epitopes using fractionated anticollagen IgG

Affinity-purified anticollagen IgG was fractionated on purified cyanogen bromide-derived collagen peptide Sepharose. The antibody fraction bound to the peptides was eluted and tested for its ability to induce passive arthritis in recipients. Anticollagen IgG bound to peptide 5 (alpha 1(II)-CB8-10 and alpha 1(II)CB11-8) and to peptide 6 (alpha 1(II)CB11) were active in inducing passive arthritis. Other peptide bound fractions were inactive. These observations suggest that the arthritogenic domain in Type II collagen is restricted to alpha 1(II)CB11.     

Bovine type I collagen: A study of cross-linking in various mature tissues

The cyanogen bromide peptides from insoluble and pepsin solubilised type I collagen of bovine bone, dentine, meniscus, tendon, skin and cornea were compared by SDS-polyacrylamide gel electrophoresis. In each case alpha 1CB6 was shown to be the only peptide of molecular weight greater than 10 000 involved in cross-linking. The major helical peptides alpha 1CB3, alpha 1CB8, alpha 1CB7 and alpha 2CB4 were not implicated in cross-linking in any tissue either by end overlap or helix-helix interaction. The C-terminal alpha 2 chain peptide alpha 2CB3,5, which contains a large helical region, was not involved in cross-linking to any large peptides, although a slight increase in molecular weight in all tissues examined did suggest a possible interaction(s) with a very small peptide of molecular weight 4--5000.     

Covalent structure of collagen: amino acid sequence of five consecutive CNBr peptides from type III collagen of human liver

Type III collagen was solubilized from human liver by limited pepsin digestion and purified by differential salt precipitation and carboxymethylcellulose chromatography. Digestion with cyanogen bromide yielded the nine distinct peptides previously described and an additional tripeptide not recognized in earlier studies. Five of these peptides, alpha1 (III)-CB1, 2, 4, 8, and 10, were further purified by molecular sieve and/or ion exchange chromatography. They contained 12, 40, 149, 125 and 3 amino acid residues, respectively. The amino acid sequence of these peptides was determined by automated Edman degradation of tryptic (before and after maleylation), chymotryptic, thermolytic or hydroxylamine-derived peptide fragments as well as the intact peptides. The alignment of these five peptides within the collagen chain is deduced to be 1-8-10-2-4 by homology with known alpha1 (I) sequences. The known CNBr peptide alignment of the NH2-terminal portion of type III collagen so far would, therefore, be alpha1 (III)-CB3-7-6-1-8-10-2-4 and correspond to the homologous region of alpha1 (I)-CB0-1-2-4-5-8-3 or residues 11-567 of the alpha1 (III) collagen chain.     

Expression, in cartilage, of a 7-amino-acid deletion in type II collagen from two unrelated individuals with Kniest dysplasia.

Kniest dysplasia is a heritable chondrodysplasia that severely affects skeletal growth. Recent evidence suggests that the etiology is based on mutations in COL2A1, the gene for collagen type II. We report the detection and partial characterization of an identical defect in type II collagen in two unrelated patients with Kniest dysplasia. Analysis of cyanogen bromide (CB)-digested cartilage samples from both probands by SDS-PAGE revealed an abnormal band for peptide alpha 1(II)CB12. The peptide was purified and digested with endoproteinase Asp-N. Fragments unique to the Kniest tissues were identified by reverse-phase high-pressure liquid chromatography and by sequence analysis. The results established a deletion of amino acids 102-108 of the alpha 1(II) triple-helical domain, which disrupted the (gly-X-Y)n repeat needed for helix formation. This was confirmed by sequence analysis of DNA amplified from both probands, revealing the molecular basis to be a single nucleotide mutation at a CpG dinucleotide (GCG-->GTG) in the codon for alanine 102. The mutation created a new splice donor site, which would account for the absence of the last seven amino acids from the 3' end of exon 12 in alpha 1(II)CB12. Light and electron micrographs of the probands' cartilage showed the perilacunar foamy matrix ("Swiss cheese") characteristic of Kniest dysplasia and chondrocytes containing dilated rough endoplasmic reticulum, which earlier studies had shown were filled with type II procollagen. These two cases strengthen the concept that Kniest dysplasia is based on mutations of COL2A1 and belongs within the broad spectrum of chondrodysplasias caused by type II collagenopathies.     

Isolation and characterization of the cyanogen bromide peptides from the B fragment of diphtheria toxin

Homogeneous fragment B, obtained through nicking of diphtheria toxin with insoluble trypsin, was cleaved with cyanogen bromide in 70% formic acid. After citraconylation, the cleavage products were separated by gel filtration on Sephadex G--75 and purified by gel filtration, ion-exchange and thin-layer or paper chromatography. Six CNBr peptides were characterized, the composition of which account for the total amino acid content of fragment B. Their apparent molecular weights are: CB 1, 12 000; CB 2, 14 000; CB 3, 8000; CB 4a, 2400; CB 4b, 2200; CB 5, 2200. CB 4a is the NH2--terminal peptide; it contains the cysteine residue of the disulfide bridge linking fragment B to fragment A. CB 3 is the COOH--terminal peptide; it bears the disulfide bridge of fragment B. Characterization of two CNBr--derived overlapping peptides provided the positioning of CB 4b and CB 2 and allowed an alignment of the CNBr peptides of fragment B to be proposed.     

Collagen cross-links: location of pyridinoline in type I collagen

Collagen from bone, dentine and tendon (type I), all of which contain the pyridinoline cross-link at varying levels, were each digested with CNBr. The resulting peptide mixtures were resolved by gel filtration on A1.5m agarose and assayed for pyridinoline. The polymeric cross-linked peptide complex, poly alpha 1CB6 [(1980) Biochem. J. 189, 111] isolated from each of these tissues did not contain pyridinoline. Only one peptide fraction contained the pyridinoline cross-link; that identified as alpha 2CB3,5. However, this peptide showed only a small increase in Mr in its cross-linked form (approx. 2000-5000) demonstrating that pyridinoline is not involved in the formation of polymeric structures like poly alpha 1CB6. These data, considered in the light of the recent finding that pyridinoline is present in type I collagens from different sources in widely varying amounts, cast doubt on its role in collagen maturation.     

Lethal perinatal osteogenesis imperfecta due to the substitution of arginine for glycine at residue 391 of the alpha 1(I) chain of type I collagen

A baby with the lethal perinatal form of osteogenesis imperfecta was shown to have a structural defect in the alpha 1(I) chain of type I procollagen. Normal and mutant alpha 1(I) CB8 cyanogen bromide peptides, from the helical part of the alpha 1(I) chains, were purified from bone. Amino acid sequencing of tryptic peptides derived from the mutant alpha 1(I) CB8 peptide showed that the glycine residue at position 391 of the alpha 1(I) chain had been replaced by an arginine residue. This substitution accounted for the more basic charged form of this peptide that was observed on two-dimensional electrophoresis of the collagen peptides obtained from the tissues. The substitution was associated with increased enzymatic hydroxylation of lysine residues in the alpha 1(I) CB8 and the adjoining CB3 peptides but not in the carboxyl-terminal CB6 and CB7 peptides. This finding suggested that the sequence abnormality had interfered with the propagation of the triple helix across the mutant region. The abnormal collagen was not incorporated into the more insoluble fraction of bone collagen. The baby appeared to be heterozygous for the sequence abnormality and as the parents did not show any evidence of the defect it is likely that the baby had a new mutation of one allele of the pro-alpha 1(I) gene. The amino acid substitution could result from a single nucleotide mutation in the codon GGC (glycine) to produce the codon CGC (arginine).     

Covalent structure of collagen: amino acid sequence of cyanogen bromide peptides from the amino-terminal segment of type III collagen of human liver

Human liver type III collagen was prepared by limited pepsin digestion, differential salt precipitation, and carboxymethylcellulose chromatography. Cyanogen bromide digestion of purified type III collagen chains yielded nine distinct peptides. Three peptides, alpha1(III)-CB3, alpha1(III)-CB7, and alpha1(III)-CB6, were isolated by carboxymethylcellulose chromatography and Sephadex G-50 SF gel filtration. Automated Edman degradation together with selective hydroxylamine cleavage and chymotrypsin and trypsin digestion enabled determination of their complete amino acid sequence. Compared with type I collagen, the data show tentative homology of alpha1(III)-CB3 with alpha1(I)-CB1, alpha1(I)-CB2, and alpha1(I)-CB4; alpha1(III)-CB7 with alpha1(I)-CB5; and alpha1(III)-CB6 with the amino-terminal portion of alpha1(I)-CB8. Close interspecies homology was found between the sequences presented here with 90 residues of alpha1(III)-CB3 and 26 of alpha1(III)-CB8 of calf aorta. The present study establishes the amino acid sequence of 229 residues near the amino terminus or nearly one-quarter of the type III collagen chains. The disaccharide, Glc-Gal, was convalently bound to hydroxylysine at a position corresponding to the same location in the alpha1(I) chain.     

Incorporation of type I collagen molecules that contain a mutant alpha 2(I) chain (Gly580-->Asp) into bone matrix in a lethal case of osteogenesis imperfecta.

To understand more directly the tissue defect in osteogenesis imperfecta (OI), bone matrix was analyzed from an infant with lethal OI (type II) of defined mutation (collagen alpha 2(I)Gly580-->Asp). Pepsin-solubilized alpha 1(I) and alpha 2(I) chains and derived CNBr-peptides migrated more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis compared with normal human controls. The peptide alpha 2(I)CB3,5, predicted to contain the mutation site, ran as a retarded doublet band and was purified by high performance liquid chromatography and digested with V8 protease. Two peptides with amino-terminal sequences beginning at residue 576 of the alpha 2(I) chain were isolated. One had the normal sequence. The other differed in that aspartic acid replaced glycine at residue 580 as predicted from cDNA analysis, and in having an unhydroxylated proline at residue 579. From yields on microsequencing and the relative intensities of the two forms of alpha 2(I)CB3,5 on SDS-polyacrylamide gel electrophoresis, the ratio of mutant to normal alpha 2(I) chains in the infant's bone matrix was 0.7/1. Although the effects of an efficient incorporation of mutant chains on the properties of the bone matrix are unknown, it may be that in this OI case the tissue abnormalities result more from the presence of mutant protein than from an underexpression of matrix.     

Covalent structure of collagen: amino acid sequence of three cyanogen bromide-derived peptides from human alpha 1(V) collagen chain

Type V collagen was prepared from human amnionic/chorionic membranes and separated into alpha 1(V) and alpha 2(V) polypeptide chains. The alpha 1(V) chain was digested with cyanogen bromide and nine peptides were obtained and purified. Three of the peptides, alpha 1(V)CB1, CB4, and CB7 having molecular weights of 5000, 8000, and 6000, respectively, were further analyzed by amino acid sequence analysis and thermolytic or tryptic digestions. CB1 contained 54 amino acids and identification of its complete sequence was aided by thermolysin digestion and isolation of two peptides, Th1 and Th2. CB4 contained 81 amino acids and sequence analysis of intact CB4 and five tryptic peptides provided us with its complete amino acid sequence. The peptide CB7 contained 67 amino acids and was cleaved into four tryptic peptides that were used for complete sequence analysis. The above results represent the first available covalent structure information on the alpha 1(V) collagen chain. These data enabled us to establish the location of these peptides within the helical structure of other collagen chains. CB4 was homologous to residues 66-145 in the collagen chain while CB1 represented residues 146-200 and CB7 was homologous with residues 201-269. This alignment was facilitated by identification of a helical collagen crossing site consisting of Hyl-Gly-His-Arg located at positions 87-90 in all collagen chains of this size thus far identified. Seventy-one percent homology (excluding Gly residues) was found between amino acids in this region of the alpha 1(XI) and of alpha 1(V) collagen chains while only 21 and 19% identity was calculated for the same region of alpha 2(V) and alpha 1(I) collagen chains, respectively.     

Conformational analysis and stability of collagen peptides by CD and by 1H- and 13C-NMR spectroscopies

Four small type I collagen CNBr peptides containing complete natural sequences were purified from bovine skin and investigated by CD and 1H- and 13C-nmr spectroscopies to obtain information concerning their conformation and thermal stability. CD showed that a triple helix was formed at 10 degrees C in acidic aqueous solution by peptide alpha l(I) CB2 only, and to lesser extent, by alpha 1(I) CB4, whereas peptides alpha 1(I) CB5 and alpha 2(I) CB2 remained unstructured. Analytical gel filtration confirmed that peptides alpha 1(I) CB2 and alpha 1(I) CB4 only were able to form trimeric species at temperature between 14 and 20 degrees C, and indicated that the monomer = trimer equilibrium was influenced by the chaotropic nature of the salt present in the eluent, by its concentration, and by temperature variations. CD measurements at increasing temperatures showed that alpha 1(I) CB2 was less stable than its synthetic counterpart due to incomplete prolyl hydroxylation of the preparation from the natural source. 1H- and 13C-nmr spectra acquired in the temperature range 0-47 and 0-27 degrees C, respectively, indicated that with decreasing temperature the most abundant from of alpha 1(I) CB2 was in slow exchange with an assembled form, characterized by broad lines, as expected for the triple-helical conformation. A large number of trimer cross peaks was observed both in the proton and carbon spectra, and these were most likely due to the nonequivalence of the environments of the three chains in the triple helix. This nonequivalence may have implications for the aggregation of collagen molecules and for collagen binding to other molecules. The thermal transition from trimer to monomer was also monitored by 1H-nmr following the change in area of the signal belonging to one of the two beta protons of the C-terminal homoserine. The unfolding process was found to be fully reversible with a melting temperature of 13.4 degrees C, in agreement with CD results. The qualitative superposition of the melting curves obtained by CD for the peptide bond characteristics and by nmr for a side chain suggests that triple-helical backbone and side chains constitute a single unit.     

Isolation and characterization of a 14-kDa albumin-binding fragment of streptococcal protein G

Protein G, a streptococcal cell wall protein, has separate binding sites for human albumin and IgG. Streptococci expressing protein G were treated with the bacteriolytic agent mutanolysin. Several IgG- and human serum albumin (HSA)-binding peptides were identified in the material thus solubilized and one of these, a 14-kDa peptide, was found to bind HSA but not IgG in Western blot experiments. This molecule was purified by affinity chromatography on Sepharose coupled with HSA followed by gel filtration on Sepharose 6B and a final affinity chromatography on IgG-Sepharose, by which low Mr W(15 to 20 kDa)IgG-binding peptides were removed. In different binding experiments the purified 14-kDa peptide bound exclusively HSA and the equilibrium constant between the peptide and HSA was determined to be 3.4 X 10(8) M-1. The relation between the 14-kDa molecule and protein G was studied by analyzing the N-terminal amino acid sequence of the peptide and comparing it with the previously determined protein G sequence. The 40 N-terminal amino acids were found to be identical with an amino acid sequence starting at position 62 in the protein G molecule. These and previous data enabled us to locate the albumin binding to the repetitively arranged domains in the N-terminal half of the protein G molecule.