Folding kinetics of the protein pectate lyase C reveal fast-forming intermediates and slow proline isomerization.
Folding kinetics of the protein pectate lyase C reveal fast-forming intermediates and slow proline isomerization.
复制标题
蛋白质果胶酸裂解酶 C 的折叠动力学揭示了快速形成的中间体和缓慢的脯氨酸异构化。
DOI：
10.1021/bi0115129
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发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Woody,RobertW
中科院分区：
文献类型：
--
作者：
Kamen,DouglasE;Woody,RobertW
果胶酸裂解酶C（Pectate lyase C，pelC）是具有平行β-螺旋折叠基序的蛋白质类的成员。本文对动力学折叠机理进行了研究。动力学圆二色性（CD）和荧光已被用来观察pelC的结构作为折叠和展开时的时间函数的变化。用远紫外CD观察到三个折叠阶段，用近紫外CD观察到四个阶段。两个最慢的相在水性缓冲液中的弛豫时间为21和46秒的顺序。双跳重折叠实验和测得的活化能谱（慢相分别为16.0和21.2 kcal/mol）表明，这两个相是脯氨酰-肽键缓慢顺反异构化的结果。我们已经确定，最早观察到的折叠阶段涉及的弛豫时间为0.25秒的大部分，如果不是全部，二级结构的形成。我们还观察到一个相位的近紫外CD上的顺序为0.25秒。这表明，沿着二级结构的出现，也产生了一些三级接触。在近紫外CD和荧光中观察到一个动力学相，其没有相应的远紫外CD相。这发生在1.1 s的弛豫时间。的折叠速率常数的自然对数的温度依赖性表明，折叠发生通过一个顺序的机制，其中一个快速平衡与未折叠的蛋白质存在的途径中间体。半经验CD计算支持pelC的β-螺旋区域在快速动力学阶段形成的想法，在该区域产生接近天然的二级和三级结构。随后是连接β-螺旋的各条链的环区域的较慢形成。
Pectate lyase C (pelC) is a member of the class of proteins that possess a parallel β-helix folding motif. A study of the kinetic folding mechanism is presented in this report. Kinetic circular dichroism (CD) and fluorescence have been used to observe changes in the structure of pelC as a function of time upon folding and unfolding. Three folding phases are observed with far-UV CD and four phases are observed with near-UV CD. The two slowest phases have relaxation times on the order of 21 and 46 s in aqueous buffer. Double-jump refolding assays and the measured activation enthalpies (16.0 and 21.2 kcal/mol for the respective slow phases) suggest that these two phases are the result of the slowcis−transisomerization of prolyl-peptide bonds. We have determined that the earliest observed folding phase involves the formation of most, if not all, of the secondary structure with a relaxation time of 0.25 s. We also observed a phase by near-UV CD on the order of 0.25 s. This suggests that along with the appearance of secondary structure, some tertiary contacts are made. There is one kinetic phase observed in the near-UV CD and fluorescence that has no corresponding far-UV CD phase. This occurs with a relaxation time of 1.1 s. The temperature dependence of the natural log of the folding rate constant suggests that folding occurs via a sequential mechanism in which an on-pathway intermediate in rapid equilibrium with the unfolded protein is present. Semiempirical CD calculations support the idea that the β-helix region of pelC forms in the fast kinetic phase, yielding near-native secondary and tertiary structures in that region. This is followed by the slower formation of the loop regions connecting individual strands of the β-helix.