Protein evolution proceeds by a complex response of organismal fitness to mutations that can simultaneously affect protein stability, structure, and enzymatic activity. To probe the relationship between genotype and phenotype, we chose a fundamental paradigm for protein evolution, folding, and design, the (βα)₈ TIM barrel fold. Here, we demonstrate the role of long‐range allosteric interactions in the adaptation of an essential hyperthermophilic TIM barrel enzyme to mesophilic conditions in a yeast host. Beneficial fitness effects observed with single and double mutations of the canonical βα‐hairpin clamps and the α‐helical shell distal to the active site revealed an underlying energy network between opposite faces of the cylindrical β‐barrel. We experimentally determined the fitness of multiple mutants in the energetic phase plane, contrasting the energy barrier of the chemical reaction and the folding free energy of the protein. For the system studied, the reaction energy barrier was the primary determinant of organism fitness. Our observations of long‐range epistatic interactions uncovered an allosteric pathway in an ancient and ubiquitous enzyme that may provide a novel way of designing proteins with a desired activity and stability profile.

中文翻译：

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变构途径解释了酵母中必需的TIM桶酶中长距离突变的有益适应性。

蛋白质进化是通过有机适应性对突变的复杂反应来进行的，这些突变可以同时影响蛋白质的稳定性，结构和酶活性。为了探究基因型和表型之间的关系，我们选择了蛋白质进化，折叠和设计的基本范式（βα）₈TIM桶折。在这里，我们证明了远程变构相互作用在必需的超嗜热TIM桶酶适应酵母宿主中嗜温条件中的作用。规范的βα-发夹夹和活动位点远端的α-螺旋壳的单突变和双突变观察到的有益健身效果揭示了圆柱形β-桶相对面之间的潜在能量网络。我们通过实验确定了高能相平面中多个突变体的适应性，对比了化学反应的能垒和蛋白质的折叠自由能。对于所研究的系统，反应能垒是机体适应性的主要决定因素。