Exploring the molecular linkage of protein stability traits for enzyme optimization by iterative truncation and evolution.
Exploring the molecular linkage of protein stability traits for enzyme optimization by iterative truncation and evolution.
复制标题
通过迭代截断和进化探索蛋白质稳定性特征的分子联系以优化酶
作者：
K.M. Einsle;Müller
蛋白质的稳定性对于它们的治疗和工业用途是至关重要的，因此是蛋白质工程的主要任务。需要几种类型的化学和物理稳定性，讨论围绕是否需要单独处理每个稳定性性状以及具体和兼容的稳定突变如何发挥作用。我们展示了一种逐步的扰动补偿策略，该策略识别突变以挽救截断的透射电子显微镜β-内酰胺酶的活性。将结构应力与热、变性剂和蛋白酶的外部应力联系起来的分析表明，我们的第二位点抑制因子是一般的稳定中心，也可以改善全长的酶。对被15个N端和3个C端残基截短的内酰胺酶变异体文库(BLA-N、Δ、15C、Δ3)进行活性选择和DNA改组。在体内表现最好的克隆包含8个突变，在Tm中超过全长野生型蛋白5.3℃，在高温下表现出显著更高的催化活性，并显示出延迟的胍诱导的变性。该突变体的晶体结构被确定，并为其稳定性决定因素提供了洞察力。N-末端和C-末端的逐步重组依次提高了其耐热性、耐变性和耐蛋白水解性，导致全长酶的aTmin值提高了15.3℃，半变性浓度从0.53 M转移到1.75M。这些改进表明，迭代截断-优化循环可以利用蛋白质中的稳定性-特性联系，特别适合于创建逐渐稳定的变体和/或缩小大小的蛋白质，而不需要详细的结构或机械信息。
The stability of proteins is paramount for their therapeutic and industrial use and, thus, is a major task for protein engineering. Several types of chemical and physical stabilities are desired, and discussion revolves around whether each stability trait needs to be addressed separately and how specific and compatible stabilizing mutations act. We demonstrate a stepwise perturbation–compensation strategy, which identifies mutations rescuing the activity of a truncated TEM β-lactamase. Analyses relating structural stress with the external stresses of heat, denaturants, and proteases reveal our second-site suppressors as general stability centers that also improve the full-length enzyme. A library of lactamase variants truncated by 15 N-terminal and three C-terminal residues (Bla-NΔ15CΔ3) was subjected to activity selection and DNA shuffling. The resulting clone with the best in vivo performance harbored eight mutations, surpassed the full-length wild-type protein by 5.3 °C inTm, displayed significantly higher catalytic activity at elevated temperatures, and showed delayed guanidine-induced denaturation. The crystal structure of this mutant was determined and provided insights into its stability determinants. Stepwise reconstitution of the N- and C-termini increased its thermal, denaturant, and proteolytic resistance successively, leading to a full-length enzyme with aTmincreased by 15.3 °C and a half-denaturation concentration shifted from 0.53 to 1.75 M guanidinium relative to that of the wild type. These improvements demonstrate that iterative truncation–optimization cycles can exploit stability–trait linkages in proteins and are exceptionally suited for the creation of progressively stabilized variants and/or downsized proteins without the need for detailed structural or mechanistic information.
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TEM-1 β-内酰胺酶的二次耐药突变可抑制错误折叠和聚集。
DOI：
10.1073/pnas.98.1.283
发表时间：
2001
影响因子：
11.1
作者：
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作者：
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影响因子：
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DOI：
--
发表时间：
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期刊：
影响因子：
--
作者：
J. Joo;Y. Yoo
通讯作者：
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