Assembly of collagen fibrils de novo by cleavage of the type I pC-collagen with procollagen C-proteinase. Assay of critical concentration demonstrates that collagen self-assembly is a classical example of an entropy-driven process.
Type I procollagen was purified from the medium of cultured human fibroblasts incubated with 14C-labeled amino acids, the NH2-terminal propeptides were cleaved with procollagen N-proteinase, and the resulting pC-collagen was isolated by gel filtration chromatography. pC-collagen did not assemble into fibrils or large aggregates even at concentrations of 0.5 mg.ml-1 at 34 degrees C in a physiological buffer. However, cleavage of pC-collagen to collagen with purified C-proteinase (Hojima, Y., (1985) J. Biol. Chem. 260, 15996-16003) generated fibrils that were visible by eye and that were large enough to be separated from solution by centrifugation at 13,000 x g for 4 min. With high concentrations of enzyme, the pC-collagen was completely cleaved in 1 h, and turbidity was near maximal in 3 h, but collagen continued to be incorporated in fibrils for over 10 h. Because the pC-collagen was uniformly labeled with 14C-aminoacids, the concentration of soluble collagen and, therefore, the critical concentration of polymerization were determined directly. The critical concentration was independent of the initial pC-collagen concentration and of the rate of cleavage. The critical concentration decreased with temperature between 29 and 41 degrees C and was 0.12 +/- 0.06 (S.E.) microgram.ml-1 at 41 degrees C. The thermodynamic parameters of assembly were essentially independent of temperature in the range 29 to 41 degrees C. The process was endothermic with a delta H value of +56 kcal.mol-1, but entropy driven with a delta S value of +220 cal.K-1.mol-1. The Gibbs energy change for polymerization was -13 kcal.mol-1 at 37 degrees C. The data demonstrate, for the first time, that type I collagen fibril formation de novo is a classical example of an entropy-driven self-assembly process similar to the polymerization of actin, flagella, and tobacco mosaic virus protein.
Kadler
K E
KE
Department of Biochemistry and Molecular Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Hojima
Y
Y
Prockop
D J
DJ
D000596:Amino Acids / Q000378:metabolism; D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins; D002850:Chromatography, Gel; D003094:Collagen / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D008433:Mathematics; D008666:Metalloendopeptidases; D011108:Polymers / Q000378:metabolism; D013816:Thermodynamics
D000596:Amino Acids; D019485:Bone Morphogenetic Proteins; D046911:Macromolecular Substances; D011108:Polymers; D003094:Collagen; D010450:Endopeptidases; D008666:Metalloendopeptidases; C525705:BMP1 protein, human; D055395:Bone Morphogenetic Protein 1