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首页> 外文期刊>Journal of Molecular Biology >Directed evolution of the DnaK chaperone: mutations in the lid domain result in enhanced chaperone activity.
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Directed evolution of the DnaK chaperone: mutations in the lid domain result in enhanced chaperone activity.

机译：DnaK伴侣的定向进化：盖子域中的突变导致伴侣活性增强。

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We improved the DnaK molecular chaperone system for increased folding efficiency towards two target proteins, by using a multi-parameter screening procedure. First, we used a folding-deficient C-terminal truncated chloramphenicol acetyl transferase (CAT_Cd9) to obtain tunable selective pressure for enhanced DnaK chaperon function in vivo. Second, we screened selected clones in vitro for CAT_Cd9 activity after growth under selective pressure. We then analyzed how these variants performed as compared to wild type DnaK towards folding assistance of a second target protein; namely, chemically denatured firefly luciferase. A total of 11 single point DnaK mutants and 1 truncated variant were identified using CAT_Cd9 as the protein target, while 4 of the 12 selected variants showed improved luciferase refolding in vitro. This shows that improving the DnaK chaperone by using a certain target substrate protein, does not necessarily result in a loss or reduction in its ability to assist other proteins. Of the 12 identified mutations, half were clustered in the nucleotide binding domain, and half in the lid domain (LD) of DnaK. The truncated variant is characterized by a 35-residue C-terminal truncation (Cd35) and exhibited the highest improvement for luciferase refolding. Cd35 showed a 7-fold increase in initial refolding rate for denatured luciferase and resulted in a 5-fold increase in maximal luminescence as compared to wild type DnaK. Given that the best in vitro performing mutants contained LD substitutions, and that the LD is not involved in ATP binding, ATP hydrolysis or client protein association, but is involved in allosteric regulation of the chaperone cycle, we propose that improved DnaK variants result in changes to allosteric domain communication, ultimately retuning the ATP-dependent chaperone cycle.
机译：我们通过使用多参数筛选程序改进了DnaK分子伴侣系统，以提高对两种靶蛋白的折叠效率。首先，我们使用了折叠缺陷型C末端截短的氯霉素乙酰转移酶（CAT_Cd9），以获得可调节的选择性压力，以增强体内DnaK伴侣功能。第二，我们在选择性压力下生长后，针对CAT_Cd9活性在体外筛选了选定的克隆。然后，我们分析了与野生型DnaK相比，这些变体在第二个靶蛋白折叠辅助下的表现。即，化学变性的萤火虫荧光素酶。使用CAT_Cd9作为蛋白质靶标，共鉴定了11个单点DnaK突变体和1个截短的变体，而12个选定变体中的4个在体外显示出改善的萤光素酶重折叠。这表明通过使用某些靶底物蛋白来改善DnaK分子伴侣，并不一定导致其辅助其他蛋白的能力的丧失或降低。在12个已识别的突变中，一半聚集在DnaK的核苷酸结合结构域中，一半聚集在盖结构域（LD）中。截短的变体的特征是具有35个残基的C端截短（Cd35），并显示出萤光素酶重新折叠的最高改进。与野生型DnaK相比，Cd35对变性的荧光素酶的初始重折叠率提高了7倍，并且最大发光强度提高了5倍。鉴于体外表现最好的突变体包含LD取代，并且LD不参与ATP结合，ATP水解或客户蛋白质缔合，但参与伴侣分子周期的变构调节，我们建议改进的DnaK变异体会导致改变到变构域通讯，最终重新调整了ATP依赖的伴侣周期。

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