The Role of Cis‐Trans Isomerization of Peptide Bonds in the Coil ⇄ Triple Helix Conversion of Collagen

Abstract

The collagen‐like portion of a peptide which comprises the amino‐terminal precursor‐specific region of bovine type III procollagen, showed an unusually fast, concentration independent and fully reversible triple helix ⇄ coil transition. A set of three interchain disulfide bridges probably provides an effective nucleus for triple helix formation. Refolding occurred in two kinetic phases. The first one was not resolved (half time < 5 s) and the second one had a half time of about 90 s at 20 °C. When measurements were performed in phosphate buffer pH 7.0, the amplitudes of both phases were of comparable size. Activation energies for the rate constant of the slow phase ranging from 44kJ/mol at 40–30 °C to 70 kJ/mol at 15–5 °C were observed. In the presence of 4 M guanidine‐HCI, essentially no fast phase was found and the activation energy was 97 ± 13 kJ/mol. The occurrence of two kinetic phases was explained by a model mechanism in which a stretch of helix between the nucleus and the first cis peptide bond is formed quickly. In the following slow phase, cis‐trans isomerization at the junction of a triple helical to coiled region becomes rate‐limiting. This interpretation was supported by temperature double‐jump experiments. The reformation of cis peptide bonds, after a fast destruction of the triple helix, was paralleled by an increase in the amplitude of the slow phase. These findings qualitatively agree with results on the role of cis‐trans isomerization in the folding of ribonuclease [Brandts, J. F., Halvorson, H. R. & Brennan, M. (1975) Biochemistry, 14, 4953–4963]. The observed curved Arrhenius plot was quantitatively explained by the normal activation energy of cis‐trans isomerization (85 kJ/mol) and the contribution of the negative enthalpy of the pre‐equilibria between partially helical species formed in the fast phase. Our data suggest that for long collagen molecules, the fast phase is negligible and cis‐trans isomerization sets an upper limit for the rate of triple helix formation in vivo. At 37 °C the minimum time estimated for complete folding is in the range of minutes and comparable with the rate of biosynthesis. At low temperatures the slow rate of cis‐trans isomerization may govern the intracellular formation of native collagen molecules to a greater extent, if no other mechanisms maintain the trans configuration of peptide bonds after release of the chains from the ribosomes. Copyright © 1978, Wiley Blackwell. All rights reserved