Triple-helix formation on ribosome-bound nascent chains of procollagen: deuterium-hydrogen exchange studies.

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Abstract
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]hydroxproline and high hydroxyproline/proline ratios. The polyribosomes were suspended in D2O at 15.degree. C and the IR 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.degree. C in the IR cells, the N-D amide II absorbance at 1480 cm-1 increased markedly, indicating that H .fwdarw. D exchange had occurred. Collagen-depleted polyribosomes showed no such changes in absorbance at 1450-1480 cm-1 on heating. Polyribosomes recovered from the infrared cells after treatment at 44.degree. C and cooling still contained collagen, as indicated by their [3H]hydroxyproline content. Nascent collagen bound to the polyribosomes can apparently assume a H-bonded structure. Taken with prior data showing that the nascent collegen 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.