A cis-prolyl peptide bond isomerization dominates the folding of the alpha subunit of Trp synthase, a TIM barrel protein.
A cis-prolyl peptide bond isomerization dominates the folding of the alpha subunit of Trp synthase, a TIM barrel protein.
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顺式脯氨酰肽键异构化主导 TRP 合酶（一种 TIM 桶蛋白）α 亚基的折叠。
DOI：
10.1016/s0022-2836(02)00737-4
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发表时间：
2002
影响因子：
5.6
通讯作者：
Matthews,CRobert
中科院分区：
文献类型：
--
作者：
Wu,Ying;Matthews,CRobert
脯氨酸肽键的顺式/反式异构化被认为是控制大肠杆菌色氨酸合成酶(αTS)α亚基折叠的主要因素。为了验证Asp27和Pro28之间独特的顺式异构体的作用，以尿素为变性剂，研究了P28A、P28G和Asp27和Gly28之间的三甘氨酸插入突变体G3P28G的折叠特性。圆二色谱分析表明，虽然P28A和P28G的芳香族侧链堆积发生了变化，但这些突变都没有显著扰乱二级结构。这三种突变蛋白都继承了野生型αTS中观察到的三态热力学行为，确保了能量表面的基本特征完好无损。动力学研究表明，丙氨酸和甘氨酸在Pro28位的取代都不会导致任何缓慢复性相的消除。相比之下，G3P28G突变体消除了最快的缓慢复性阶段和两个展开阶段之一。G3P28G上的双跳实验证实了缺失的复性相归因于Asp27-Pro28肽键的异构化。这些结果表明，含有顺式肽键的紧密、重叠的转角所传达的局部稳定性足以有利于几个非脯氨基残基的顺式异构体。驱动异构化反应所需的自由能由稳定中间体的形成提供，这表明在αTS的折叠过程中，需要获得结构和稳定性来诱导后续的限速步骤。
The cis/trans isomerization of prolyl peptide bonds has been suggested to dominate the folding of the alpha subunit of tryptophan synthase from Escherichia coli (αTS). To test the role of the unique cis isomer between Asp27 and Pro28, the folding properties of P28A, P28G and G3P28G, a three-glycine insertion mutant between Asp27 and Gly28, were investigated using urea as a denaturant. Circular dichroism analysis demonstrated that none of the mutations perturb the secondary structure significantly, although the aromatic side-chain packing is altered for P28A and P28G. All three mutant proteins inherited the three-state thermodynamic behavior observed in wild-type αTS, ensuring that the fundamental features of the energy surface are intact. Kinetic studies showed that neither alanine nor glycine substitutions at Pro28 results in the elimination of any slow-refolding phases. By contrast, the G3P28G mutant eliminates the fastest of the slow-refolding phases and one of the two unfolding phases. Double-jump experiments on G3P28G confirm the assignment of the missing refolding phase to the isomerization of the Asp27-Pro28 peptide bond. These results imply that the local stability conveyed by the tight, overlapping turns containing the cis peptide bond is sufficient to favor the cis isomer for several non-prolyl residues. The free energy required to drive the isomerization reaction is provided by the formation of the stable intermediate, demonstrating that the acquisition of structure and stability is required to induce subsequent rate-limiting steps in the folding of αTS.
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影响因子：
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通讯作者：
T. Tsuji;B. Chrunyk;X. Chen;C. Matthews
影响因子：
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作者：
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