4-Thiaproline accelerates the slow folding phase of proteins containing cis prolines in the native state by two orders of magnitude.
4-Thiaproline accelerates the slow folding phase of proteins containing cis prolines in the native state by two orders of magnitude.
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DOI：
10.1002/pro.4877
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发表时间：
2024-02
期刊：
影响因子：
8
通讯作者：
Rubini, Marina
中科院分区：
文献类型：
--
作者：
Loughlin, Jennie O';Zinovjev, Kirill;Napolitano, Silvia;van Der Kamp, Marc;Rubini, Marina
肽基-脯氨酸肽键的顺式/反式异构化通常是天然状态下含有顺式脯氨酸残基的蛋白质重折叠反应的瓶颈。脯氨酸（Pro）类似物，特别是C4取代的氟脯氨酸，已广泛应用于蛋白质工程中，以提高多肽和蛋白质的热力学稳定性，并研究折叠动力学。4‐硫脯氨酸（Thp）已被证明对Pro的环皱褶有偏倚，与Pro相比，增加了模型肽的顺式居群百分比，并降低了顺式/反式异构化反应的活化能垒。尽管Thp具有有趣的特性，但它很少被结合到蛋白质中。此外，Thp对球状蛋白折叠动力学的影响从未被报道过。在这项研究中，我们发现在cisPro76中加入Thp到硫氧还蛋白变体Trx1P后，重折叠反应的半衰期从~2小时缩短到~35秒。用Thp替代cisPro48后，伪野生型barstar蛋白的重折叠速率也显著加快。量子化学计算表明，吡咯烷环上的c γ - h2基团被硫原子取代，可能由于肽键的减弱而降低顺/反式旋转的势垒。在加入Thp后，蛋白质变体保持了其热力学稳定性，而修饰后的Trx1P的催化和酶活性保持不变。我们的研究结果表明，Pro isostee Thp可能会加速含有顺式脯氨酸残基的蛋白质在天然状态下的缓慢再折叠反应，而不依赖于局部结构环境。
The cis/trans isomerization of peptidyl‐prolyl peptide bonds is often the bottleneck of the refolding reaction for proteins containing cis proline residues in the native state. Proline (Pro) analogues, especially C4‐substituted fluoroprolines, have been widely used in protein engineering to enhance the thermodynamic stability of peptides and proteins and to investigate folding kinetics. 4‐thiaproline (Thp) has been shown to bias the ring pucker of Pro, to increase the cis population percentage of model peptides in comparison to Pro, and to diminish the activation energy barrier for the cis/trans isomerization reaction. Despite its intriguing properties, Thp has been seldom incorporated into proteins. Moreover, the impact of Thp on the folding kinetics of globular proteins has never been reported. In this study, we show that upon incorporation of Thp at cisPro76 into the thioredoxin variant Trx1P the half‐life of the refolding reaction decreased from ~2 h to ~35 s. A dramatic acceleration of the refolding rate could be observed also for the protein pseudo wild‐type barstar upon replacement of cisPro48 with Thp. Quantum chemical calculations suggested that the replacement of the CγH2 group by a sulfur atom in the pyrrolidine ring, might lower the barrier for cis/trans rotation due to a weakened peptide bond. The protein variants retained their thermodynamic stability upon incorporation of Thp, while the catalytic and enzymatic activities of the modified Trx1P remained unchanged. Our results show that the Pro isostere Thp might accelerate the rate of the slow refolding reaction for proteins containing cis proline residues in the native state, independent from the local structural environment.
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