Adenylate kinase (AK) orthologs with a range of thermostabilities were subjected to random circular permutation, and deep mutational scanning was used to evaluate where new protein termini were nondisruptive to activity. The fraction of circularly permuted variants that retained function in each library correlated with AK thermostability. In addition, analysis of the positional tolerance to new termini, which increase local conformational flexibility, showed that bonds were either functionally sensitive to cleavage across all homologs, differentially sensitive, or uniformly tolerant. The mobile AMP-binding domain, which displays the highest calculated contact energies, presented the greatest tolerance to new termini across all AKs. In contrast, retention of function in the lid and core domains was more dependent upon AK melting temperature. These results show that family permutation profiling identifies primary structure that has been selected by evolution for dynamics that are critical to activity within an enzyme family. These findings also illustrate how deep mutational scanning can be applied to protein homologs in parallel to differentiate how topology, stability, and local energetics govern mutational tolerance.

中文翻译：

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蛋白质对随机圆形排列的耐受性与残基-残基接触的热稳定性和局部能量学相关。


对具有一系列热稳定性的腺苷酸激酶 (AK) 直系同源物进行随机循环排列，并使用深度突变扫描来评估新蛋白质末端在何处不破坏活性。每个文库中保留功能的循环排列变体的比例与 AK 热稳定性相关。此外，对新末端的位置耐受性（增加局部构象灵活性）的分析表明，键对所有同系物的裂解功能敏感、差异敏感或一致耐受。移动 AMP 结合域显示出计算出的最高接触能，在所有 AK 中对新末端具有最大的耐受性。相比之下，盖子和核心区域功能的保留更依赖于 AK 熔化温度。这些结果表明，家族排列分析识别了进化所选择的一级结构，以实现对酶家族内的活性至关重要的动力学。这些发现还说明了如何将深度突变扫描并行应用于蛋白质同源物，以区分拓扑结构、稳定性和局部能量学如何控制突变耐受性。