Protein Assembly of Procollagen and Effects of Hydroxylation

Abstract

The three polypeptide chains of a precursor of collagen, procollagen, were found to join their NH,-terminal regions in disulfide linkage independently of the state of folding of the remainder of the polypeptide chains.This folding becomes altered from random coil to collagen helix by secondary modification, hydroxylation, of the chains.When hydroxylation was inhibited with cr , a'-dipyridyl, chick embryo skull bones synthesized and accumulated such disulfide-linked procollagen.Ultracentrifugal velocity sedimentation analysis of this radioactively labeled material at different temperatures showed that it was in a random coil configuration at chick body temperature: T, for denaturation was 25".If hydroxylation was restored, by adding ferric ions, then the previously synthesized procollagen became folded.This was deduced from velocity sedimentation analyses in different concentrations of the denaturant, urea, at constant temperature.Subsequently to hydroxylation the procollagen was proteolytically cleaved, producing collagen in the same sequence as material which was synthesized in the absence of CY, cu'-dipyridyl.The T, of normally hydroxylated procollagen was found to be 42O.Therefore hydroxylation of procollagen leads to collagen helix formation at chick body temperature.A technique of differential sedimentation analysis of radioactive macromolecules is described which showed a consistent difference in the sedimentation of hydroxylated and nonhydroxylated procollagens, whether as native triple-stranded molecules, or as denatured triplet chains held together by disulfide linkage, or as single, denatured chains after reduction.This difference approximately corresponds to the difference in mass which accompanies hydroxylation and glycosylation.The disulfide-linked triplet procollagen which was synthesized while hydroxylation was inhibited with CY, a!-dipyridyl was shown to contain two polypeptide chains of type pro-al and one pro-a2 per molecule.Therefore the portions of each procollagen chain which are additional to the collagen sequence, and which have been called "registration peptides," are able to associate selectively to triplets under conditions which preclude collagen helix formation.