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首页> 外文期刊>Journal of Molecular Biology >Parallel channels and rate-limiting steps in complex protein folding reactions: prolyl isomerization and the alpha subunit of Trp synthase, a TIM barrel protein.
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Parallel channels and rate-limiting steps in complex protein folding reactions: prolyl isomerization and the alpha subunit of Trp synthase, a TIM barrel protein.

机译：复杂蛋白质折叠反应中的平行通道和限速步骤：脯氨酰异构化和TIM桶蛋白Trp合酶的α亚基。

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A kinetic folding mechanism for the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, involving four parallel channels with multiple native, intermediate and unfolded forms, has recently been proposed. The hypothesis that cis/trans isomerization of several Xaa-Pro peptide bonds is the source of the multiple folding channels was tested by measuring the sensitivity of the three rate-limiting phases (tau(1), tau(2), tau(3)) to catalysis by cyclophilin, a peptidyl-prolyl isomerase. Although the absence of catalysis for the tau(1) (fast) phase leaves its assignment ambiguous, our previous mutational analysis demonstrated its connection to the unique cis peptide bond preceding proline 28. The acceleration of the tau(2) (medium) and tau(3) (slow) refolding phases by cyclophilin demonstrated that cis/trans prolyl isomerization is also the source of these phases. A collection of proline mutants, which covered all of the remaining 18 trans proline residues of alphaTS, was constructed to obtain specific assignments for these phases. Almost all of the mutant proteins retained the complex equilibrium and kinetic folding properties of wild-type alphaTS; only the P217A, P217G and P261A mutations caused significant changes in the equilibrium free energy surface. Both the P78A and P96A mutations selectively eliminated the tau(1) folding phase, while the P217M and P261A mutations eliminated the tau(2) and tau(3) folding phases, respectively. The redundant assignment of the tau(1) phase to Pro28, Pro78 and Pro96 may reflect their mutual interactions in non-random structure in the unfolded state. The non-native cis isomers for Pro217 and Pro261 may destabilize an autonomous C-terminal folding unit, thereby giving rise to kinetically distinct unfolded forms. The nature of the preceding amino acid, the solvent exposure, or the participation in specific elements of secondary structure in the native state, in general, are not determinative of the proline residues whose isomerization reactions can limit folding.
机译：最近已经提出了来自大肠杆菌的色氨酸合酶（αTS）的α亚基的动力学折叠机制，该机制涉及具有四个天然，中间和未折叠形式的四个平行通道。通过测量三个限速相（tau（1），tau（2），tau（3）的敏感性，检验了多个Xaa-Pro肽键的顺式/反式异构化是多个折叠通道来源的假设。 ）被亲环蛋白（一种肽基脯氨酰异构酶）催化。尽管没有对tau（1）（快速）相进行催化使它的分配模棱两可，但我们先前的突变分析表明它与脯氨酸28之前的独特顺式肽键有关。tau（2）（中等）和tau的加速作用（3）亲环蛋白的（缓慢）重折叠相表明顺式/反式脯氨酰异构化也是这些相的来源。构建了涵盖αTS其余所有18个反式脯氨酸残基的脯氨酸突变体的集合，以获得这些阶段的特定分配。几乎所有的突变蛋白都保留了野生型alphaTS的复杂平衡和动力学折叠特性。仅P217A，P217G和P261A突变引起平衡自由能表面的显着变化。 P78A和P96A突变都选择性地消除了tau（1）折叠相，而P217M和P261A突变分别消除了tau（2）和tau（3）折叠相。将tau（1）阶段分配给Pro28，Pro78和Pro96可能会在展开状态下以非随机结构反映它们的相互影响。 Pro217和Pro261的非天然顺式异构体可能会破坏自动C末端折叠单元的稳定性，从而产生动力学上不同的未折叠形式。通常，前述氨基酸的性质，溶剂暴露或天然状态下二级结构的特定元素的参与并不能确定其异构化反应可限制折叠的脯氨酸残基。

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