Structural Aspects of Hydroxyproline-Containing Proteins

Abstract

The occurrence of hydroxyproline (Hyp) in collagen, C1q and acetylcholineesterase (AChE) raises important questions concerning the role of this unusual imino acid in the structure and function of these proteins. Available data on collagen indicate that Hyp is necessary for the normal secretion of the protein after its synthesis and for the integrity of the triple-helical conformation. Studies from our laboratory have dealt with the structural aspects of the posttranslational conversion of proline to hydroxyproline in collagen mediated by prolyl hydroxylase. We proposed that the beta-turn conformation at the Pro-Gly segments in the nascent procollagen molecule are the sites of the enzymatic hydroxylation and that this conformation changes over to the collagen-like helix as a result of the hydroxylation process. Recently, we have provided additional experimental support to our proposal by a) synthesizing specific beta-turn oligopeptides containing the Pro-Gly as well as Pro-Ala and Pro-DAla sequences and showing that these act as inhibitors of the enzymatic hydroxylation of a synthetic substrate and b) demonstrating, by circular dichroism spectroscopy, the occurrence of a conformational change leading to the triple-helix as a direct consequence of proline hydroxylation in a non-helical polypeptide substrate. We have also observed that the acquisition of hydroxylation results in a significant enhancement of the rate of folding of the polypeptide chain from the unfolded to the triple-helical conformation. We believe that our observations on proline hydroxylation in collagen should also be applicable to C1q and acetylcholineesterase both of which share the general structural and functional properties of collagen in their "tail" regions. Using the techniques employed in collagen studies, one should be able to assess the role of hydroxyproline in the folding, structural stabilities and functions of C1q and AChE. This would also involve the study of the unhydroxylated and hydroxylated precursors of these proteins which may share common structural features with their collagen counterparts. Finally, a systematic study of hydroxyproline-containing peptides and polypeptides has been initiated by us so as to understand the exact manner in which Hyp participates in the formation and stability of the triple-helical conformation in the proteins in which it occurs.

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“…Hydroxylation of proline, resulting in hydroxyproline (Hyp), is a posttranslational modification best characterized in collagen, a major protein in all vertebrates ( , ). Hydroxylation of proline in the Pro-Hyp-Gly repeats stabilizes the collagen triple helix, although the mechanism by which the hydroxyl group contributes to protein stability is not fully understood ( − ).…”

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“…It is known for a long time that l-ascorbic acid, better known as vitamin C, is required for collagen protein synthesis [48][49][50], for example by being the co-factor for prolyl hydroxylase, an enzyme that converts proline residues of the α-chains of collagen into hydroxyproline [51,52]. This amino acid stabilizes the triple helix; a decreased prolyl hydroxyation of the α-chains results in unstable collagen molecules [52,53]. Such molecules are degraded intracellularly and are not transported out of the cell.…”