Evolutionary Analysis As a Powerful Complement to Energy Calculations for Protein Stabilization

Stability is one of the most important characteristics of proteins employed as biocatalysts, biotherapeutics, and biomaterials, and the role of computational approaches in modifying protein stability is rapidly expanding. We have recently identified stabilizing mutations in haloalkane dehalogenase DhaA using phylogenetic analysis but were not able to reproduce the effects of these mutations using force-field calculations. Here we tested four different hypotheses to explain the molecular basis of stabilization using structural, biochemical, biophysical, and computational analyses. We demonstrate that stabilization of DhaA by the mutations identified using the phylogenetic analysis is driven by both entropy and enthalpy contributions, in contrast to primarily enthalpy-driven stabilization by mutations designed by the force-field calculations. Comprehensive bioinformatics analysis revealed that more than half (53%) of 1 099 evolution-based stabilizing mutations would be evaluated as destabilizing by force-field calculations. Thermodynamic integration considers both folded and unfolded states and can describe the entropic component of stabilization, yet it is not suitable for predictive purposes due to its high computational demands. Altogether, our results strongly suggest that energetic calculations should be complemented by a phylogenetic analysis in protein-stabilization endeavors.作为生物催化剂、生物治疗药物及生物材料使用的蛋白质，稳定性是其最为核心的特性之一，而计算方法在改造蛋白质稳定性领域的作用正快速拓展。我们此前通过系统发育分析（phylogenetic analysis）在卤烷脱卤酶DhaA（haloalkane dehalogenase DhaA）中鉴定得到可提升稳定性的突变位点，但无法通过力场计算（force-field calculations）复现这些突变所带来的效应。本研究针对该稳定化效应的分子机制提出四种不同假说，并通过结构生物学、生物化学、生物物理及计算生物学分析手段进行验证。我们证实，经系统发育分析鉴定得到的突变体对DhaA的稳定作用同时由熵（entropy）与焓（enthalpy）的贡献共同驱动，与力场计算设计得到的突变体所介导的、主要以焓驱动的稳定效应形成鲜明对比。全面的生物信息学分析显示，在1099个基于进化的稳定性提升突变体中，超过半数（53%）会被力场计算判定为去稳定化突变。热力学积分（thermodynamic integration）可同时考虑蛋白质的折叠态与去折叠态，能够刻画稳定化效应的熵组分，但由于其极高的计算资源需求，并不适用于预测性应用。综上，我们的研究结果强烈表明，在蛋白质稳定化改造相关研究中，应将能量计算与系统发育分析相结合。