In vitro collagen fibril assembly in glycerol solution: evidence for a helical cooperative mechanism involving microfibrils.
Glycerol inhibits the in vitro self-association of monomeric collagen into fibrils and induces the dissociation of fibrils preassembled from NaBH4-reduced collagen. These effects were investigated in an effort to understand the mechanism of fibril assembly of the protein. In PS buffer (0.03 M NaPi and 0.1 M NaCl, pH 7.0) containing 0.1-1.0 M glycerol, the self-association of type I collagen from calf skin took place only if the protein concentration was above a critical value. This critical protein concentration increased with increasing glycerol concentration. Velocity sedimentation studies showed that below the critical protein concentration and under fibril assembly conditions, the collagen was predominantly in a monomeric state. Electron microscopic examinations revealed that the collagen aggregates formed above the critical concentration consisted mostly of microfibrils of 3-5-nm diameter along with some banded fibrils were found. Collagen treated with pepsin to remove its nonhelical telopeptides also self-associated into microfibrils and fibrils in the presence of glycerol, but the reaction did not exhibit any critical concentration. These results are consistent with a mechanism of in vitro collagen fibril assembly which involves the initial formation of microfibrils through a helical cooperative mechanism. They also suggest that contacts of the nonhelical telopeptides of each collagen with its neighboring molecules provide the necessary negative free energy change for the cooperativity and that subsequent lateral association of the microfibrils leads to banded fibrils.
D000818:Animals; D001894:Borohydrides; D002417:Cattle; D003094:Collagen / Q000378:metabolism*; D005990:Glycerol / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008854:Microscopy, Electron; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012867:Skin / Q000378:metabolism; D014443:Tyrosine / Q000032:analysis
D001894:Borohydrides; D046911:Macromolecular Substances; D014443:Tyrosine; D003094:Collagen; D005990:Glycerol