Designing functional proteins that can withstand extreme heat is beneficial for industrial and protein therapeutic applications. Thus, elucidating the atomic-level determinants of thermostability is a major interest for rational protein design. To that end, we compared the structure and dynamics of a set of previously designed, thermostable proteins based on the activation domain of human procarboxypeptidase A2 (AYEwt). The mutations in these designed proteins were intended to increase hydrophobic core packing and inter-secondary-structure interactions. To evaluate whether these design strategies were successfully deployed, we performed all-atom, explicit-solvent molecular dynamics (MD) simulations of AYEwt and three designed variants at both 25 and 100°C. Our MD simulations agreed with the relative experimental stabilities of the designs based on their secondary structure content, Cα root-mean-square deviation/fluctuation, and buried-residue solvent accessible surface area. Using a contact analysis, we found that the designs stabilize inter-secondary structure interactions and buried hydrophobic surface area, as intended. Based on our analysis, we designed three additional variants to test the role of helix stabilization, core packing, and a Phe → Met mutation on thermostability. We performed the additional MD simulations and analysis on these variants, and these data supported our predictions.

中文翻译：

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分子动力学模拟表明，从头设计的α/β蛋白具有稳定的突变。

设计可以承受极高热量的功能蛋白对于工业和蛋白质治疗应用是有益的。因此，阐明热稳定性的原子水平决定因素是合理蛋白质设计的主要兴趣。为此，我们根据人羧肽酶A2（AYEwt）的激活域，比较了一组先前设计的热稳定蛋白的结构和动力学。这些设计蛋白中的突变旨在增加疏水性核心堆积和二级结构间的相互作用。为了评估这些设计策略是否成功部署，我们在25和100°C下对AYEwt和三种设计的变体进行了全原子，显式溶剂分子动力学（MD）模拟。我们的MD模拟基于其二级结构含量，Cα均方根偏差/波动和掩埋残留溶剂可及表面积，与设计的相对实验稳定性相符。使用接触分析，我们发现设计可以稳定二级结构间的相互作用和掩埋的疏水表面积。根据我们的分析，我们设计了三个其他变体，以测试螺旋稳定性，核心堆积和Phe→Met突变对热稳定性的作用。我们对这些变体进行了附加的MD模拟和分析，这些数据支持了我们的预测。我们发现设计可以稳定二级结构间的相互作用，并掩埋疏水表面积。根据我们的分析，我们设计了三个其他变体来测试螺旋稳定性，核心堆积和Phe→Met突变对热稳定性的作用。我们对这些变体进行了附加的MD模拟和分析，这些数据支持了我们的预测。我们发现设计可以稳定二级结构间的相互作用，并掩埋疏水表面积。根据我们的分析，我们设计了三个其他变体来测试螺旋稳定性，核心堆积和Phe→Met突变对热稳定性的作用。我们对这些变体进行了附加的MD模拟和分析，这些数据支持了我们的预测。