Nucleation and propagation of the collagen triple helix in single-chain and trimerized peptides:: Transition from third to first order kinetics
Nucleation and propagation of the collagen triple helix in single-chain and trimerized peptides:: Transition from third to first order kinetics
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DOI：
10.1006/jmbi.2002.5439
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发表时间：
2002-03-29
影响因子：
5.6
通讯作者：
Engel, J
中科院分区：
文献类型：
--
作者：
Boudko, S;Frank, S;Engel, J
研究了胶原模型（ProProGly）（10）和（ProHypGly）（10）在宽浓度范围内从单个非交联肽链形成三螺旋的动力学，并与成核三聚体中的动力学进行了比较。在非常低的肽浓度下，反应级数为3，但在较高浓度下降低。对于（ProProGly）10，三级速率常数在7 ℃下为800 M（-2）s（-1），这对应于在60 μ M链浓度下15小时的非常长的半衰期。对于（ProHypGly）10，速率常数高约1000倍，这与4-羟脯氨酸在胶原中的稳定作用一致。的反应顺序的浓度依赖性解释的成核机制，其中一个非常不稳定的二聚体是在快速平衡的单体链和添加的第三链发生在一个限速步骤。在高浓度下，成核比螺旋形成的传播快，并且传播变得限速。为了检验这一假设，通过将（ProProGly）10与T4噬菌体的三聚体折叠结构域或胶原蛋白III的交联结构域GlyProGlyProCysCysGlyGlyGly融合来引入人工核。将这些结构域重组连接至（GlyProPro）的C末端（10），并以与胶原蛋白III中的C末端前肽结构域相似的方式连接三条链。这导致约1 M的局部固有链浓度。观察到（GlyProPro）（10）折叠子中三螺旋折叠的一级反应，半衰期为8.3分钟，这与1 M肽浓度下单链折叠的速率大致匹配。发现该反应的高活化能为54 kJ/mol，而成核步骤的温度依赖性接近于零，证实了天然胶原蛋白的早期发现，即肽键的顺-反异构化是传播中的限速步骤。(C)2002 Elsevier Science Ltd.
The kinetics of triple helix formation from single non-crosslinked peptide chains were studied for the collagen models (ProProGly)(10) and (ProHypGly)(10) in a broad concentration range and compared with those in nucleated trimers. At very low peptide concentrations the reaction order is 3 but decreases at higher concentrations. For (ProProGly)10 the third order rate constant is 800 M(-2) s(-1) at 7degreesC, which corresponds to a very long half time of 15 hours at 60 muM chain concentration. For (ProHypGly)10 the rate constant is about 1000-fold higher, which is consistent with the stabilizing effect of 4-hydroxyproline in collagens. The concentration dependence of the reaction order is explained by a nucleation mechanism in which a very unstable dimer is in fast equilibrium with the monomeric chains and addition of the third chain occurs in a rate-limiting step. At high concentrations nucleation is faster than propagation of helix formation and propagation becomes rate-limiting. To test this hypothesis an artificial nucleus was introduced by fusion of (ProProGly)10 with the trimeric foldon domain of T4 phage or the crosslinking domain of collagen III GlyProProGlyProCysCysGlyGlyGly. These domains were recombinantly attached to the C terminus of (GlyProPro)(10) and link the three chains in a similar way to the C-terminal propeptide domain in collagen III. This results in a local intrinsic chain concentration of about 1 M. A first order reaction is observed for the folding of the triple helix in (GlyProPro)(10)foldon with a half time of 8.3 minutes, which approximately matches the rate of folding from single chains at 1 M peptide concentration. A high activation energy of 54 kJ/mol is found for this reaction, whereas the temperature dependence of the nucleation step is close to zero, confirming earlier findings on natural collagens that cis-trans isomerization of peptide bonds is the rate-limiting step in propagation. (C) 2002 Elsevier Science Ltd.