Collagen triple-helix formation in all-trans chains proceeds by a nucleation/growth mechanism with a purely entropic barrier
Collagen triple-helix formation in all-trans chains proceeds by a nucleation/growth mechanism with a purely entropic barrier
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DOI：
10.1073/pnas.0505141102
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发表时间：
2005-09-27
影响因子：
11.1
通讯作者：
Bächinger, HP
中科院分区：
文献类型：
--
作者：
Bachmann, A;Kiefhaber, T;Bächinger, HP
胶原蛋白由重复的Gly-Xaa-Yaa三肽单元组成，其中脯氨酸和羟脯氨酸分别常见于Xaa和Yaa位置。该序列基序允许形成高度规则的三螺旋，其通过组成聚脯氨酸-II-螺旋中的空间（熵）限制和三条链之间的骨架氢键来稳定。浓度依赖性缔合反应和缓慢脯氨酰异构化步骤已被确定为胶原蛋白折叠过程中的主要限速过程。为了获得在没有这些缓慢反应的情况下三螺旋形成的动力学信息，我们对来自人III型胶原的交联片段进行了停流双跳实验。这种技术使我们能够测量浓度无关的折叠动力学从展开的链与所有peparticle键的反式构象。结果表明，在3.7摄氏度下，三螺旋形成的速率常数为113 +/- 20 s（-1），并且几乎与温度无关，表明纯粹的熵势垒。比较胍氯对折叠动力学和稳定性的影响表明，限速步骤是通过将10个连续的三肽单元（每条链3.3个）带入三螺旋构象来表示的。随后加入的三肽单位发生在一个更快的时间尺度上，不能观察到实验。这些结果支持胶原三螺旋形成的熵控制拉链样成核/生长机制。
Collagen consists of repetitive Gly-Xaa-Yaa tripepticle units with proline and hydroxyproline frequently found in the Xaa and Yaa position, respectively. This sequence motif allows the formation of a highly regular triple helix that is stabilized by steric (entropic) restrictions in the constituent polyproline-II-helices and backbone hydrogen bonds between the three strands. Concentration-dependent association reactions and slow prolyl isomerization steps have been identified as major rate-limiting processes during collagen folding. To gain information on the dynamics of triple-helix formation in the absence of these slow reactions, we performed stopped-flow double-jump experiments on cross-linked fragments derived from human type III collagen. This technique allowed us to measure concentration-independent folding kinetics starting from unfolded chains with all pepticle bonds in the trans conformation. The results show that triple-helix formation occurs with a rate constant of 113 +/- 20 s(-1) at 3.7 degrees C and is virtually independent of temperature, indicating a purely entropic barrier. Comparison of the effect of guaniclinium chloride on folding kinetics and stability reveals that the rate-limiting step is represented by bringing 10 consecutive tripepticle units (3.3 per strand) into a triple-helical conformation. The following addition of tripepticle units occurs on a much faster time scale and cannot be observed experimentally. These results support an entropy-controlled zipper-like nucleation/growth mechanism for collagen triple-helix formation.