Triple-helix formation on ribosome-bound nascent chains of procollagen: deuterium-hydrogen exchange studies.
Polyribosomes containing nascent [3H]proline-labeled collagen chains were isolated from chick embryo fibroblasts in culture. These nascent chains were nearly completely hydroxylated, as indicated by the presence of [3H]hydroxyproline and high hydroxyproline/proline ratios. The polyribosomes were suspended in D2O at 15 degrees and the infrared spectrum was determined using a reference cell containing collagen-depleted polyribosomes in D2O, matched to equal RNA content. The amide I and amide II bands were observed. When the polyribosomes were heated in D2O at 44 degrees in the infrared cells, the N--D amide II absorbance at 1480 cm-1 increased markedly, indicating that H leads to D exchange had occurred. Collagen-depleted polyribosomes showed no such changes in absorbance at 1450-1480 cm-1 upon heating. Polyribosomes recovered from the infrared cells after treatment at 44 degrees and cooling still contained collagen, as indicated by their [3H]hydroxyproline content. These data indicate that nascent collagen bound to the polyribosomes can assume a hydrogen-bonded structure. Taken with prior data showing that the nascent collagen was also resistant to pepsin digestion, it is suggested that the collagen examined is in triple-helix conformation. Because the nascent polyribosome-bound collagen is nearly fully hydroxylated, it must be considered that triple-helix formation can occur between nascent chains while they are attached to the endoplasmic reticulum surface and that chain association and triple-helix formation in vivo may well occur before rather than after release.
D000818:Animals; D002478:Cells, Cultured; D003903:Deuterium; D005347:Fibroblasts; D006860:Hydrogen Bonding; D006909:Hydroxyproline / Q000378:metabolism; D011132:Polyribosomes / Q000378:metabolism*; D011347:Procollagen / Q000096:biosynthesis; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared
D011347:Procollagen; D003903:Deuterium; D006909:Hydroxyproline
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