Stability and activity of mesophilic subtilisin E and its thermophilic homolog: Insights from molecular dynamics simulations
Stability and activity of mesophilic subtilisin E and its thermophilic homolog: Insights from molecular dynamics simulations
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DOI：
10.1021/ja990420s
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发表时间：
1999-07-28
影响因子：
15
通讯作者：
Merz, KM
中科院分区：
文献类型：
--
作者：
Colombo, G;Merz, KM
在这里，我们研究了嗜热突变酶（标记为5-3H5）高温（350 K）行为的起源，参见Zhao和Arnold Prot。工程学报，1999,12,47-53)由枯草菌素E经8个氨基酸取代而得到。通过使用分子动力学（MD）模拟，我们提供了点突变如何影响蛋白质结构和动力学的分子水平见解。从我们的模拟中，我们观察到几个关键区域的均势标准差降低，整体柔韧性增加，氢键数量增加，亲热系统中稳定相互作用的数量增加。我们还表明，在低温下，嗜热酶并不一定比它们的亲中温酶更不灵活。然而，为了保持活性，嗜热酶必须在高温下保持其三维结构和柔韧性。此外，我们已经能够指出一些单一取代的影响。即使还不可能给出合理蛋白质设计的一般规则，我们也能够对蛋白质应该如何稳定以使其具有嗜热性做出一些预测。特别地，我们建议加速热稳定蛋白设计的一个有希望的策略是通过使用MD模拟（或适当的实验）来识别蛋白质内的流动区域。据推测，这些区域允许自催化反应发生，也参与允许水进入蛋白质内部并启动蛋白质展开。这些流动区域也可能对催化装置的定位产生不利影响，从而降低催化效率。因此，一旦这些位置被确定，“集中”定向进化研究就可以被设计来稳定这些“流动”区域。
Herein we examine the origin of the high-temperature(350 K) behavior of a thermophilic mutant enzyme (labeled as 5-3H5; see Zhao and Arnold Prot. Eng. 1999, 12, 47-53) derived from subtilisin E by eight amino acid substitutions. Through the use of molecular dynamics (MD) simulations, we have provided molecular-level insights into how point mutations can affect protein structure and dynamics. From our simulations we observed a reduced rmsd in several key regions, an increased overall flexibility, an increase in the number of hydrogen bonds, and an increase in the number of stabilizing interactions in the thermophilic system. We also show that it is not a necessary requirement that thermophilic enzymes be less flexible than their mesophilic counterparts at low temperatures. However, thermophilic enzymes must retain their three-dimensional structures and flexibility at high temperatures in order to retain activity. Furthermore, we have been able to point out the effects of some of the single substitutions. Even if ii is not possible yet to give general rules for rational protein design, we are able to make some predictions on how a protein should be stabilized in order to be thermophilic. In particular, we suggest that a promising strategy toward speeding up the design of thermally stable proteins would be to identify fluxional regions within a. protein through the use of MD simulations (or suitable experiments). Presumably these regions allow for autocatalytic reactions to occur and are also involved in allowing water to gain access to the interior of the protein and initiate protein unfolding. These fluxional regions could also adversely affect the positioning of the catalytic machinery, thereby decreasing catalytic efficiency. Thus, once these locations have been identified, "focused" directed evolution studies could be designed that stabilize these "fluxional" regions.