Folding mechanism of the triple helix in type-III collagen and type-III pN-collagen. Role of disulfide bridges and peptide bond isomerization.
The kinetics of triple-helix formation in type III pN-collagen, type III collagen and a quarter fragment of type III collagen was followed by optical rotation and circular dichroism. Kinetic intermediates were detected by trypsin digestion and polyacrylamide gel electrophoresis. The end products of refolding at 25 degrees C were identical to the native molecules according to their melting profiles, molecular weights and sedimentation behavior. Only at low temperatures (4-15 degrees C) were mismatched structures of lower stability formed. At 25 degrees C helix formation started exclusively at the set of three disulfide bridges which link the three chains at the carboxy-terminal end. The growth of the triple helix proceeds from this single nucleus at a rather uniform rate in a zipper-like fashion. This gives rise to zero-order kinetics over a large fraction of the conversion. Consequently the time of half conversion is proportional to the length of the molecule. From the appearance and disappearance of intermediates the growth of the triple helix could be observed directly. The rate of helix propagation is determined by the rate of cis leads to trans isomerization of peptide bonds. A model mechanism was devloped which quantitatively described the overall kinetics as well as the time course of the intermediates with a single set of parameters: the rate constant of cis leads to trans isomerization k = 0.015 s-1 and an average number of 30 tripeptide units in uninterrupted stretches of residues with all peptide bonds in trans configuration.
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003094:Collagen; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D007536:Isomerism; D007700:Kinetics; D008956:Models, Chemical; D008970:Molecular Weight; D009904:Optical Rotation; D011487:Protein Conformation; D013696:Temperature
D004220:Disulfides; D003094:Collagen