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首页> 外文期刊>Journal of Molecular Biology >Protein dynamics in a family of laboratory evolved thermophilic enzymes.
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Protein dynamics in a family of laboratory evolved thermophilic enzymes.

机译：实验室进化的嗜热酶家族中的蛋白质动力学。

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Molecular dynamics simulations were employed to study how protein solution structure and dynamics are affected by adaptation to high temperature. Simulations were carried out on a para-nitrobenzyl esterase (484 residues) and two thermostable variants that were generated by laboratory evolution. Although these variants display much higher melting temperatures than wild-type (up to 18 degrees C higher) they are both >97% identical in sequence to the wild-type. In simulations at 300K the thermostable variants remain closer to their crystal structures than wild-type. However, they also display increased fluctuations about their time-averaged structures. Additionally, both variants show a small but significant increase in radius of gyration relative to wild-type. The vibrational density of states was calculated for each of the esterases. While the density of states profiles are similar overall, both thermostable mutants show increased populations of the very lowest frequency modes (<10cm(-1)), with the morestable mutant showing the larger increase. This indicates that the thermally stable variants experience increased concerted motions relative to wild-type. Taken together, these data suggest that adaptation for high temperature stability has resulted in a restriction of large deviations from the native state and a corresponding increase in smaller scale fluctuations about the native state. These fluctuations contribute to entropy and hence to the stability of the native state. The largest changes in localized dynamics occur in surface loops, while other regions, particularly the active site residues, remain essentially unchanged. Several mutations, most notably L313F and H322Y in variant 8G8, are in the region showing the largest increase in fluctuations, suggesting that these mutations confer more flexibility to the loops. As a validation of our simulations, the fluctuations of Trp102 were examined in detail, and compared with Trp102 phosphorescence lifetimes that were previously measured. Consistent with expectations from the theory of phosphorescence, an inverse correlation between out-of-plane fluctuations on the picosecond time scale and phosphorescence lifetime was observed.
机译：分子动力学模拟用于研究蛋白质溶液的结构和动力学如何受高温影响。对对硝基苄基酯酶（484个残基）和由实验室进化产生的两个热稳定变异体进行了模拟。尽管这些变体显示出比野生型高得多的解链温度（最高高达18摄氏度），但它们的序列均与野生型具有> 97％的同一性。在300K的模拟中，热稳定变体比野生型更接近其晶体结构。但是，它们的平均时间结构也显示出越来越大的波动。另外，相对于野生型，两种变体均显示出旋转半径的很小但显着的增加。计算每种酯酶的状态振动密度。虽然总体状态密度相似，但两个热稳定突变体均显示出频率最低模式（<10cm（-1））的增加，而更稳定的突变体则显示出较大的增加。这表明热稳定的变体相对于野生型经历增加的协同运动。综上所述，这些数据表明对高温稳定性的适应已导致对与原始状态的大偏差的限制以及围绕原始状态的较小尺度波动的相应增加。这些波动有助于熵，从而有助于原始状态的稳定性。局部动力学的最大变化发生在表面环中，而其他区域，特别是活性位点残基，基本上保持不变。几个突变，最显着的是变体8G8中的L313F和H322Y，在该区域中波动增加最大，表明这些突变为环赋予了更大的灵活性。为了验证我们的仿真，对Trp102的波动进行了详细检查，并将其与先前测量的Trp102磷光寿命进行了比较。与磷光理论的预期一致，在皮秒时间尺度上的面外波动与磷光寿命之间存在反比关系。

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