The Role of Cis-Trans Isomerization of Peptide Bonds in the Coil ⇄ Triple Helix Conversion of Collagen
The Role of Cis-Trans Isomerization of Peptide Bonds in the Coil ⇄ Triple Helix Conversion of Collagen
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螺旋中肽键顺反异构化的作用⇄ 胶原蛋白三螺旋转化
DOI：
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发表时间：
1978
期刊：
影响因子：
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通讯作者：
R. Timpl
中科院分区：
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作者：
H. Bächinger;J. Engel;P. Bruckner;R. Timpl
包含牛III型前胶原的氨基末端双链体特异性区域的肽的胶原样部分显示出异常快速的、不依赖于浓度的且完全可逆的三螺旋双链体转变。一组三个链间二硫键可能提供了一个有效的核三螺旋形成。折叠过程分为两个动力学阶段。在20 °C下，第一个未被解析（半衰期<5s），第二个具有约90 s的半衰期。当在pH 7.0的磷酸盐缓冲液中进行测量时，两个相位的幅度具有可比性。慢相的速率常数的活化能范围为44 kJ/mol（40-30 °C）至70 kJ/mol（15-5 °C）。在4 M盐酸胍存在下，基本上没有发现快相，活化能为97 ± 13 kJ/mol。
两个动力学阶段的发生解释了一个模型机制，其中一个伸展的螺旋之间的核和第一顺式肽键迅速形成。在下面的慢相，顺反异构化的交界处的三重螺旋卷曲区域成为限速。这一解释得到了温度双跳实验的支持。在三螺旋快速破坏后，顺式肽键的重新形成被慢相振幅的增加所抑制。这些发现在定性上与顺-反异构化在核糖核酸酶折叠中的作用的结果一致[Brandts，J.F.，Halvorson，H. R. & Brennan，M.（1975）Biochemistry，14，4953-4963]。所观察到的曲线Arrhenius图定量地解释了顺反异构化的正常活化能（85 kJ/mol）和在快速相形成的部分螺旋物种之间的预平衡的负焓的贡献。我们的数据表明，对于长的胶原蛋白分子，快相是可以忽略不计的，顺反异构化设置了一个上限，在体内的三螺旋形成的速度。在37 °C下，估计完全折叠的最短时间在几分钟的范围内，并且与生物合成的速率相当。在低温下，如果没有其他机制在链从核糖体释放后维持肽键的反式构型，则顺-反异构化的缓慢速率可能在更大程度上控制天然胶原分子的细胞内形成。
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.