Does the Triple Helical Domain of Type I Collagen Encode Molecular Recognition and Fiber Assembly while Telopeptides Serve as Catalytic Domains?
1999; Elsevier BV; Volume: 274; Issue: 51 Linguagem: Inglês
10.1074/jbc.274.51.36083
ISSN1083-351X
AutoresNatalia V. Kuznetsova, Sergey Leikin,
Tópico(s)Enzyme Production and Characterization
ResumoOver the last several decades, it has been established that proteolytic removal of short, non-helical terminal peptides (telopeptides) from type I collagen significantly alters the kinetics of in vitro fibrillogenesis. However, it has also been observed that the protein is still capable of forming fibers even after complete removal of telopeptides. This study focuses on the characterization of this fibrillogenesis competency of collagen. We have combined traditional kinetic and thermodynamic assays of fibrillogenesis efficacy with direct measurements of interaction between collagen molecules in fibers by osmotic stress and x-ray diffraction. We found that telopeptide cleavage by pepsin or by up to 20 h of Pronase treatment altered fiber assembly kinetics, but the same fraction of the protein still assembled into fibers. Small-angle x-ray diffraction showed that these fibers have normal, native-like D-stagger. Force measurements indicated that collagen-collagen interactions in fibers were not affected by either pepsin or Pronase treatment. In contrast, prolonged (>20 h) Pronase treatment resulted in cleavage of the triple helical domain as indicated by SDS-polyacrylamide gel electrophoresis. The triple-helix cleavage correlated with the observed decrease in the fraction of protein capable of forming fibers and with the measured loss of attraction between helices in fibers. These data suggest that telopeptides play a catalytic role, whereas the information necessary for proper molecular recognition and fiber assembly is encoded in the triple helical domain of collagen. Over the last several decades, it has been established that proteolytic removal of short, non-helical terminal peptides (telopeptides) from type I collagen significantly alters the kinetics of in vitro fibrillogenesis. However, it has also been observed that the protein is still capable of forming fibers even after complete removal of telopeptides. This study focuses on the characterization of this fibrillogenesis competency of collagen. We have combined traditional kinetic and thermodynamic assays of fibrillogenesis efficacy with direct measurements of interaction between collagen molecules in fibers by osmotic stress and x-ray diffraction. We found that telopeptide cleavage by pepsin or by up to 20 h of Pronase treatment altered fiber assembly kinetics, but the same fraction of the protein still assembled into fibers. Small-angle x-ray diffraction showed that these fibers have normal, native-like D-stagger. Force measurements indicated that collagen-collagen interactions in fibers were not affected by either pepsin or Pronase treatment. In contrast, prolonged (>20 h) Pronase treatment resulted in cleavage of the triple helical domain as indicated by SDS-polyacrylamide gel electrophoresis. The triple-helix cleavage correlated with the observed decrease in the fraction of protein capable of forming fibers and with the measured loss of attraction between helices in fibers. These data suggest that telopeptides play a catalytic role, whereas the information necessary for proper molecular recognition and fiber assembly is encoded in the triple helical domain of collagen. acid-soluble collagen pepsin-treated collagen Pronase-treated collagen polyacrylamide gel electrophoresis polyethylene glycol Type I collagen is a major component of the extracellular matrix in all higher vertebrates, e.g. it is the main structural protein of skin, bone, and tendon (see, for example, Refs. 1Kadler K. Protein Profile. 1994; 1: 519-638PubMed Google Scholar and 2Prockop D.J. Kivirikko K.I. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1402) Google Scholar). Each molecule is a heterotrimer composed of two α1(I) chains and one α2(I) chain. It contains a long triple helical domain (∼1000 residues from each chain) and short, non-helical terminal peptides (telopeptides). In vitro, under appropriate conditions, type I molecules spontaneously form fibers that are virtually indistinguishable from native fibers by electron microscopy (see, for example, Ref. 3Williams B.R. Gelman R.A. Poppke D.C. Piez K.A. J. Biol. Chem. 1978; 253 (and references therein): 6578-6585Abstract Full Text PDF PubMed Google Scholar). X-ray diffraction patterns from such reconstituted fibers and from native fibers are similar as well (4Eikenberry E.F. Brodsky B. J. Mol. Biol. 1980; 144: 397-404Crossref PubMed Scopus (28) Google Scholar, 5Brodsky B. Eikenberry E.F. Cassidy-Belbruno K. Sterling K. Biopolymers. 1982; 21: 935-951Crossref PubMed Scopus (50) Google Scholar, 6Brodsky B. Eikenberry E.F. Methods Enzymol. 1982; 82: 127-174Crossref PubMed Scopus (115) Google Scholar, 7Brodsky B. Tanaka S. Eikenberry E.F. Nimni M.E. Collagen. I. CRC Press, Inc., Boca Raton, FL1988: 95-112Google Scholar). It is commonly believed that type I collagen contains all structural information that is necessary for its self-assembly into fibers, except maybe for some tissue-specific factors (see, for example, Ref. 8Veis A. Payne K. Nimni M.E. Collagen. I. CRC Press, Inc., Boca Raton, FL1988: 113-137Google Scholar). However, the location of the "coding" regions, the nature of this information, and how it is "translated" into intermolecular forces responsible for fibrillogenesis are still poorly understood. One of the debated issues is the role of telopeptides. Telopeptides form covalent cross-links with triple helical regions on opposing molecules (see, for example, Refs. 9Eyre D.R. Annu. Rev. Biochem. 1984; 53: 717-748Crossref PubMed Google Scholar and 10Yamauchi M. Mechanic G.L. Nimni M.E. Collagen. I. CRC Press, Inc., Boca Raton, FL1988: 157-172Google Scholar). It is believed that this occurs after completion of fibrillogenesis and that the cross-links stabilize rather than create appropriate molecular arrangement. It was also suggested that telopeptides are important at earlier stages,i.e. in the process of fibrillogenesis. This hypothesis is based primarily on studies of enzymatically treated collagen. Specifically, partial or complete removal of telopeptides alters the kinetics of collagen fiber formation (11Comper W.D. Veis A. Biopolymers. 1977; 16: 2113-2131Crossref PubMed Scopus (112) Google Scholar, 12Helseth D.L. Veis A. J. Biol. Chem. 1981; 256: 7118-7128Abstract Full Text PDF PubMed Google Scholar). It may also affect fiber morphology, as indicated by electron microscopy (11Comper W.D. Veis A. Biopolymers. 1977; 16: 2113-2131Crossref PubMed Scopus (112) Google Scholar, 13Drake M.P. Davison P.F. Bump S. Schmitt F.O. Biochemistry. 1966; 5: 301-312Crossref PubMed Scopus (140) Google Scholar, 14Leibovich S.J. Weiss J.B. Biochim. Biophys. Acta. 1970; 214: 445-454Crossref PubMed Scopus (84) Google Scholar, 15Ghosh S.K. Mitra H.P. Biochim. Biophys. Acta. 1975; 405: 340-346Crossref PubMed Scopus (11) Google Scholar, 16Gelman R.A. Poppke D.C. Piez K.A. J. Biol. Chem. 1979; 254: 11741-11745Abstract Full Text PDF PubMed Google Scholar). Additional evidence for possible telopeptide involvement at early stages of fiber assembly comes from inhibition of intact collagen fibrillogenesis by synthetic peptides that have sequences found in carboxyl-terminal telopeptides (17Prockop D.J. Fertala A. J. Biol. Chem. 1998; 273: 15598-15604Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). These data clearly establish that telopeptides play some role in fibrillogenesis kinetics. Do they mean, however, that telopeptides contain a code necessary for recognition and appropriate packing of collagen into fibers? This work revisits the question. It builds on recent advances in direct measurement of intermolecular forces by osmotic stress and x-ray diffraction (18Rau D.C. Lee B. Parsegian V.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2621-2625Crossref PubMed Scopus (386) Google Scholar, 19Parsegian V.A. Rand R.P. Fuller N.L. Rau D.C. Methods Enzymol. 1986; 127: 400-416Crossref PubMed Scopus (487) Google Scholar, 20Leikin S. Rau D.C. Parsegian V.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 276-280Crossref PubMed Scopus (137) Google Scholar, 21Leikin S. Rau D.C. Parsegian V.A. Nat. Struct. Biol. 1995; 2: 205-210Crossref PubMed Scopus (143) Google Scholar). We apply this new technique to measurement of forces between collagen molecules in fibers and combine it with traditional in vitro fibrillogenesis assays. We compare acid-soluble collagen (AcCol)1 with pepsin-treated collagen (PepCol) and with Pronase-treated collagen (PronCol). We find that marked changes in collagen-collagen interactions and in fibrillogenesis competency occur only after prolonged Pronase treatment. These changes coincide with cleavage of the triple helix rather than with telopeptide removal. Type I collagen was extracted from rat tail tendon as described previously (20Leikin S. Rau D.C. Parsegian V.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 276-280Crossref PubMed Scopus (137) Google Scholar, 21Leikin S. Rau D.C. Parsegian V.A. Nat. Struct. Biol. 1995; 2: 205-210Crossref PubMed Scopus (143) Google Scholar, 22Kuznetsova N. Chi S.L. Leikin S. Biochemistry. 1998; 37: 11888-11895Crossref PubMed Scopus (94) Google Scholar). Briefly, frozen tails of young rats were purchased from Pel-Freez Biologicals and stored frozen at −20 °C. Tails were thawed in cold (4 °C) protease-inhibiting buffer (3.5 m NaCl, 10 mm Tris, 20 mm EDTA, 2 mm N-ethylmaleimide, and 1 mm phenylmethylsulfonyl fluoride (pH 7.5)). Tendons were excised from tails, washed in the same buffer for several days at 4 °C, and then dissolved in 0.5 m acetic acid (pH 2.8). Solubilized tendons were digested by pepsin (Calbiochem) as described (23Miller E.J. Rhodes R.K. Methods Enzymol. 1982; 82: 33-64Crossref PubMed Scopus (682) Google Scholar) or by Pronase (Calbiochem) as described (13Drake M.P. Davison P.F. Bump S. Schmitt F.O. Biochemistry. 1966; 5: 301-312Crossref PubMed Scopus (140) Google Scholar). Pepsin was added directly to the solution of collagen in acetic acid at a ratio of 100 mg of pepsin/1 g of tendons in two doses for 24 h at 4 °C each. For Pronase treatment, solubilized tendons were dialyzed against 0.1m calcium acetate (pH 7.0). Pronase was added to the substrate at a ratio of 1:100 and stirred at 23 °C for 68 h. Aliquots of digested collagen were taken at 20, 44, and 68 h after the reaction was started. The reaction was stopped by addition of an equal volume of 0.5 m acetic acid. AcCol, PepCol, and PronCol were purified by three cycles of salt precipitation and acetic acid resolubilization (23Miller E.J. Rhodes R.K. Methods Enzymol. 1982; 82: 33-64Crossref PubMed Scopus (682) Google Scholar) and stored in 0.5m acetic acid at 4 °C. Samples from each preparation were characterized by SDS-polyacrylamide gel electrophoresis (PAGE) (3% stacking gel and 6% separating gel, stained by Coomassie Blue R-250). Collagen concentration in solutions was measured by Sircol assay (Accurate Chemical & Scientific Corp.) and/or by optical absorbance in the 215–230 nm region. Both assays were calibrated using a set of standard collagen solutions of different concentrations. Tyrosine content of collagen was estimated from the optical absorbance at 275.5 nm of collagen denatured in 6 m guanidine hydrochloride following the procedure proposed in Ref. 24Edelhoch H. Biochemistry. 1967; 6: 1948-1954Crossref PubMed Scopus (3052) Google Scholar. For type I collagen, it was shown previously that this spectroscopic method agrees with direct amino acid analysis within ∼10% (25Chandrakasan G. Torchia D.A. Piez K.A. J. Biol. Chem. 1976; 251: 6062-6067Abstract Full Text PDF PubMed Google Scholar, 26Na G.C. Collagen Relat. Res. 1988; 8: 315-330Crossref PubMed Scopus (45) Google Scholar). The kinetics of fiber formation and equilibrium solubility of collagen at 32 °C were measured as described (22Kuznetsova N. Chi S.L. Leikin S. Biochemistry. 1998; 37: 11888-11895Crossref PubMed Scopus (94) Google Scholar). Briefly, collagen was dialyzed against 2 mmHCl (pH 2.7). Aliquots from dialyzed collagen solutions (0.5–1.5 mg/ml) were mixed 1:1 on ice with 2× initiation buffer (20 mm sodium phosphate and 0.26 m NaCl with pH adjusted to give pH 7.4 in the mixture). The mixture was degassed for 5 min under vacuum and immediately placed into a Jasco V 560 spectrophotometer, where it was maintained at 32 °C. Fibrillogenesis kinetics was monitored by recording the optical density at 450 nm as a function of time. When no further change in the optical density was detected, the precipitate of assembled collagen fibers was spun down from the mixture by centrifugation at 14,000 × g for 5 min. Collagen solubility at 32 °C or the percentage of collagen competent to form fibers was evaluated from the protein concentration in the supernatant. The supernatant and precipitate were characterized by SDS-PAGE. Native fibers from rat tail tendons as well as fibers reconstituted from solutions of AcCol, PepCol, and PronCol were used. Native fibers were excised from rat tails, stored in the protease inhibitor buffer as described above, and then directly used for force measurement. Alternatively, fibers were transferred from the protease inhibitor buffer into 0.1 m sodium phosphate and 2m glycerol (pH 7.5), in which they were washed for several days at 4 °C and after that used for force measurement. We refer to the latter fibers as "washed native." Reconstituted collagen fibers were made by slow concentration of AcCol, PepCol, and PronCol in 0.5 m acetic acid. Each solution was dialyzed at 4 °C in a Pierce Model 500 microdialyzer system until a solid protein film was formed. Dialysis was performed against 50% polyethylene glycol (PEG; average M r 8000; U. S. Biochemical Corp.) solution in 0.5 m acetic acid. The film was further equilibrated in 40–50 weight % solution of PEG 8000 in 10 mm Tris and 2 mm EDTA (pH 7.5) for 2–3 days at 4 °C, washed in the same buffer to remove PEG, and air-dried at 4 °C. It was shown previously that such reconstituted films, prepared either from AcCol or from PepCol, consist of densely packed, native-like collagen fibers (20Leikin S. Rau D.C. Parsegian V.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 276-280Crossref PubMed Scopus (137) Google Scholar). The films were cut into small pieces (∼1 × 0.5 × 0.5 mm) and prehydrated in 10 mm Tris and 2 mm EDTA (pH 7.5) at 4 °C for at least 1 day to ensure more reproducible measurements of interaxial distances. However, this procedure resulted in the loss of integrity and, apparently, partial solubilization of films made from PronCol treated with the enzyme for 44 and 68 h. Thus, we had to skip the prehydration step for the latter samples. Forces between helices in fibers were measured as a function of interaxial distance by the osmotic stress technique and x-ray diffraction (18Rau D.C. Lee B. Parsegian V.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2621-2625Crossref PubMed Scopus (386) Google Scholar, 19Parsegian V.A. Rand R.P. Fuller N.L. Rau D.C. Methods Enzymol. 1986; 127: 400-416Crossref PubMed Scopus (487) Google Scholar, 20Leikin S. Rau D.C. Parsegian V.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 276-280Crossref PubMed Scopus (137) Google Scholar, 21Leikin S. Rau D.C. Parsegian V.A. Nat. Struct. Biol. 1995; 2: 205-210Crossref PubMed Scopus (143) Google Scholar). Native fibers or pieces of reconstituted films were equilibrated for at least 1 week in gravimetrically prepared PEG 8000 solutions (2–50 weight % PEG in 10 mm Tris and 2 mm EDTA (pH 7.5)) at 5, 20, or 35 °C in tightly sealed 1.5-ml microtubes fitted with O-rings. The solutions were refreshed in the middle of the equilibration (after the first 2–3 days). Each equilibrated sample was sealed in a specially designed cell with a small amount of the solution and placed into an FR590 x-ray diffractometer (Enraf Nonius), where it was maintained at the corresponding temperature during the measurement. This diffractometer was optimized for mid-angle x-ray diffraction (Δq ∼ 0.05-Å−1 resolution in reciprocal space). Details of the design of the x-ray equipment were as described (20Leikin S. Rau D.C. Parsegian V.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 276-280Crossref PubMed Scopus (137) Google Scholar, 21Leikin S. Rau D.C. Parsegian V.A. Nat. Struct. Biol. 1995; 2: 205-210Crossref PubMed Scopus (143) Google Scholar, 22Kuznetsova N. Chi S.L. Leikin S. Biochemistry. 1998; 37: 11888-11895Crossref PubMed Scopus (94) Google Scholar, 27Mudd C.P. Tipton H. Parsegian V.A. Rau D.C. Rev. Sci. Instrum. 1987; 58: 2110-2114Crossref Scopus (17) Google Scholar). The lowest order Bragg spacing (d Br) for lateral packing of the helices was measured, and interaxial distance (d int) was calculated from it in the approximation of hexagonal packing (d int = 2d Br/√3). PEG, because of its large size, does not penetrate inside collagen fibers, whereas water inside fibers freely exchanges with surrounding solution (20Leikin S. Rau D.C. Parsegian V.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 276-280Crossref PubMed Scopus (137) Google Scholar, 21Leikin S. Rau D.C. Parsegian V.A. Nat. Struct. Biol. 1995; 2: 205-210Crossref PubMed Scopus (143) Google Scholar, 28Kuznetsova N. Rau D.C. Parsegian V.A. Leikin S. Biophys. J. 1997; 72: 353-362Abstract Full Text PDF PubMed Scopus (50) Google Scholar). As a result, PEG osmotically compresses fibers. This action of PEG is counteracted by interaction between collagen helices in fibers that is responsible for fiber swelling. From thermodynamic analysis of this force balance, it was shown that the corresponding force between helices (f) per unit of their length is given by (18Rau D.C. Lee B. Parsegian V.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2621-2625Crossref PubMed Scopus (386) Google Scholar) f = ΠPEG d int/√3, provided that lateral packing of collagen can be approximated by a hexagonal lattice and interactions between collagen helices are pairwise additive. After force measurement, at least three samples of each collagen were reequilibrated in 6% PEG in 10 mm Tris and 2 mm EDTA (pH 7.5) and sealed in the same x-ray cell as for force measurement. The samples were exposed overnight in an Elliot GX-13 x-ray diffractometer equipped with a 100-μm focusing cup, a multilayer x-ray lens (45-cm focal length; Osmic), and a single set of slits placed immediately after the lens. Diffraction patterns were captured on CRST-VN image plates (Fuji) and read using a BAS2500 image plate scanner (Fuji). The sample-to-plate distance was ∼40 cm. The size of the x-ray beam at the focal spot on the plate was ∼200 × 320 μm. The resolution was estimated as Δq ∼ 0.003 Å−1. The lowest measurable scattering vector q was ∼0.04–0.05 Å−1. This could not be improved due to limitations associated with the design of the x-ray lens. However, such resolution was sufficient for the purpose of this work. We evaluated integrity of collagen molecules by SDS-PAGE and by estimating the number of tyrosine residues/molecule from UV absorption spectra. We assumed that rat tail tendon collagen is similar to mouse type I collagen, whose complete primary sequence is known (29Phillips C.L. Morgan A.L. Lever L.W. Wenstrup R.J. Genomics. 1992; 13: 1345-1346Crossref PubMed Scopus (24) Google Scholar, 30Li S.W. Khillan J. Prockop D.J. Matrix Biol. 1995; 14: 593-595Crossref PubMed Scopus (26) Google Scholar). Based on the mouse sequence (α1(I) chain: CA11_MOUSE, Swiss-Prot accession number P11087; and α2(I) chain: CA21_MOUSE, Swiss-Prot accession number Q01149), one expects an intact molecule to contain 14 tyrosines: 12 in telopeptides and 2 in the triple helical region of the α2(I) chain. Judging from SDS-PAGE, AcCol that did not undergo any enzymatic treatment contained α1(I) and α2(I) chains along with higher molecular weight complexes (Fig. 1,lane 1). The complexes are due to covalent cross-links between two telopeptide chains of the same molecule or between a telopeptide on one molecule and a helical domain on another molecule (9Eyre D.R. Annu. Rev. Biochem. 1984; 53: 717-748Crossref PubMed Google Scholar, 10Yamauchi M. Mechanic G.L. Nimni M.E. Collagen. I. CRC Press, Inc., Boca Raton, FL1988: 157-172Google Scholar). From UV absorption at 275 nm, the number of tyrosine residues in AcCol was estimated as ∼10 (Table I). Apparently, some AcCol molecules had damaged telopeptides.Table INumber of tyrosine residues in acid-soluble and enzymatically treated collagens estimated from UV absorption spectraCollagenEstimated average No. of Tyr residues/moleculeaThe values were obtained using a 1500m−1 cm−1 extinction coefficient for Tyr at 275.5 nm, as described (24). For type I collagen, this spectroscopic method agrees with direct amino acid analysis within ∼10% (25, 26). In our case, the most likely source of error was base-line subtraction. For AcCol, PepCol, and 20-h PronCol, the uncertainty in base-line subtraction did not exceed 20%. For 44- and 68-h PronCol, the base-line subtraction was unreliable, and only upper bounds for the tyrosine content could be estimated.AcCol10 ± 2PepCol5 ± 120-h PronCol2.3 ± 0.544-h PronCol<1.668-h PronCol<1.2a The values were obtained using a 1500m−1 cm−1 extinction coefficient for Tyr at 275.5 nm, as described (24Edelhoch H. Biochemistry. 1967; 6: 1948-1954Crossref PubMed Scopus (3052) Google Scholar). For type I collagen, this spectroscopic method agrees with direct amino acid analysis within ∼10% (25Chandrakasan G. Torchia D.A. Piez K.A. J. Biol. Chem. 1976; 251: 6062-6067Abstract Full Text PDF PubMed Google Scholar, 26Na G.C. Collagen Relat. Res. 1988; 8: 315-330Crossref PubMed Scopus (45) Google Scholar). In our case, the most likely source of error was base-line subtraction. For AcCol, PepCol, and 20-h PronCol, the uncertainty in base-line subtraction did not exceed 20%. For 44- and 68-h PronCol, the base-line subtraction was unreliable, and only upper bounds for the tyrosine content could be estimated. Open table in a new tab The pepsin treatment of collagen removed higher molecular weight complexes and reduced the molecular weight of α1(I) and α2(I) chains (Fig. 1, compare lanes 1 and 2). It is believed that this is due to partial cleavage of non-helical telopeptides (13Drake M.P. Davison P.F. Bump S. Schmitt F.O. Biochemistry. 1966; 5: 301-312Crossref PubMed Scopus (140) Google Scholar, 14Leibovich S.J. Weiss J.B. Biochim. Biophys. Acta. 1970; 214: 445-454Crossref PubMed Scopus (84) Google Scholar, 31Weiss J.B. Int. Rev. Connect. Tissue Res. 1976; 7: 101-157Crossref PubMed Google Scholar). The observed decrease in the number of tyrosines/molecule from ∼10 in AcCol to ∼5 in PepCol supports this interpretation, as previously shown (26Na G.C. Collagen Relat. Res. 1988; 8: 315-330Crossref PubMed Scopus (45) Google Scholar). After 20 h of Pronase treatment (20-h PronCol), the major α1 and α2 bands on SDS-PAGE were similar to those in PepCol (Fig. 1, comparelanes 2 and 3). From UV absorption, we found ∼2 tyrosines/20-h PronCol molecule. Most likely, these are the tyrosines located within the triple helical domain of the α2 chain. Apparently, all telopeptide tyrosines are removed by 20 h of Pronase treatment, including those at the interface between the triple helix and carboxyl-terminal telopeptides. This indicates complete telopeptide digestion, in agreement with previous reports (12Helseth D.L. Veis A. J. Biol. Chem. 1981; 256: 7118-7128Abstract Full Text PDF PubMed Google Scholar, 13Drake M.P. Davison P.F. Bump S. Schmitt F.O. Biochemistry. 1966; 5: 301-312Crossref PubMed Scopus (140) Google Scholar, 32Davison P.F. Drake M.P. Biochemistry. 1966; 5: 313-321Crossref PubMed Scopus (40) Google Scholar). In contrast to pepsin, Pronase treatment led to two additional bands that appeared on the gel close to the α1 and α2 bands. Evidently, Pronase not only digests telopeptides, but also cleaves collagen at some specific site that lies within the triple helical domain. These bands must be products of the cleavage of the α1 and α2 chains, and we designated the bands as α1′ and α2′, respectively. This is consistent with earlier observations that Pronase slowly cleaves collagen even after complete digestion of telopeptides (13Drake M.P. Davison P.F. Bump S. Schmitt F.O. Biochemistry. 1966; 5: 301-312Crossref PubMed Scopus (140) Google Scholar, 32Davison P.F. Drake M.P. Biochemistry. 1966; 5: 313-321Crossref PubMed Scopus (40) Google Scholar). Longer Pronase digestion (44 h (Fig. 1, lane 5) and 68 h (lane 7)) led to intensification of the α1′ and α2′ bands and to the appearance of two more bands labeled as α1″ and α2″. The latter bands most likely result from cleavage of the α1 and α2 chains at yet another site within the triple helical domain. The tyrosine content of collagen was further reduced to 20 h) Pronase treatment, i.e. only when digestion of triple helical domains became pronounced. After each kinetic experiment (24 h), we separated assembled fibers and the soluble fraction by centrifugation and measured collagen concentration in the soluble fraction. A significant amount of collagen may be present in this fraction either because collagen becomes assembly-incompetent (as a result of enzymatic cleavage) or because fibrillogenesis is incomplete even after 24 h (as a result of very slow kinetics). We found that virtually all AcCol and PepCol assembled into fibers, with only trace amounts remaining in the soluble fraction. Thus, the equilibrium solubility of assembly-competent collagen is negligibly small. After 20 h of Pronase treatment, the kinetics was still sufficiently fast (Fig. 2 a) so that fiber assembly was complete at the time of fraction separation and concentration measurement (24 h). Thus, in equilibrium, ∼80% of 20-h PronCol formed fibers, whereas the remaining protein stayed in solution. As indicated by SDS-PAGE, 20-h PronCol contained some molecules with cleaved triple helical regions (Fig. 1, lane 3). The soluble fraction consisted virtually only of damaged molecules that had the cleaved α1′ and α2′ chains instead of the normal α1 and α2 chains (Fig. 1, lane 4). The fraction of assembly-competent collagen decreased with the length of Pronase treatment as shown in Fig. 2 b. This coincided with enhancement of the α1′ and α2′ bands and the appearance of the α1″ and α2″ bands on SDS-PAGE of PronCol (Fig. 1, lanes 5 and 7). Long Pronase treatment (44 and 68 h) resulted in extremely slow fibrillogenesis kinetics so that the process was not complete after 24 h. Thus, although the soluble protein fraction contained primarily α1′, α1″, α2′, and α2″ chains, it also had some intact α1 and α2 chains (Fig. 1, lanes 6 and8). To assess changes in structure and interaction between collagen molecules in fibers, we prepared reconstituted protein films as described under "Materials and Methods." The structure of these films can be determined by comparing their small-angle x-ray diffraction patterns with a similar pattern from native rat tail tendons, as shown in Fig. 3. In native tendon fibers, a characteristic set of multiple reflections can be clearly seen (Fig. 3, curve a). These reflections are higher orders of diffraction arising from d = 670-Å axial periodicity in collagen fibers. The positions of maxima in theq-space are given by q(n) = 2πn/d, where n is the order of the diffraction and q is the scattering vector. Note that the 5th, 9th, 12th, 20th, and 21st orders are significantly stronger than their neighboring peaks. This pattern is a signature of the native D-stagger of collagen molecules (4Eikenberry E.F. Brodsky B. J. Mol. Biol. 1980; 144: 397-404Crossref PubMed Scopus (28) Google Scholar, 6Brodsky B. Eikenberry E.F. Methods Enzymol. 1982; 82: 127-174Crossref PubMed Scopus (115) Google Scholar, 7Brodsky B. Tanaka S. Eikenberry E.F. Nimni M.E. Collagen. I. CRC Press, Inc., Boca Raton, FL1988: 95-112Google Scholar, 33Fraser R.D.B. MacRae T.P. Conformation in Fibrous Proteins. Academic Press, New York1973Google Scholar). The small-angle diffraction pattern from reconstituted AcCol films (Fig. 3, curve b) is very similar to the pattern from native fibers in terms of both positions and relative intensities of the diffraction peaks, consistent with what was reported previously (4Eikenb
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