Systems, Synthetic, and Physical Biology Graduate Program, Rice University, 6100 Main Street, MS-180, Houston, TX 77005, USA.
Department of BioSciences, Rice University, 6100 Main Street, MS-140, Houston, TX 77005, USA.
Protein Eng Des Sel. 2019 Dec 31;32(11):489-501. doi: 10.1093/protein/gzaa012.
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.
对具有不同热稳定性的腺苷酸激酶 (AK) 同源物进行随机环状排列，并进行深度突变扫描，以评估新蛋白末端在不破坏活性的情况下所处的位置。在每个文库中，保留功能的环状排列变体的比例与 AK 的热稳定性相关。此外，对增加局部构象灵活性的新末端的位置容忍度的分析表明，在所有同源物中，键要么对切割具有功能敏感性，要么具有差异敏感性，要么具有均匀的耐受性。具有最高计算接触能的可移动 AMP 结合域在所有 AK 中对新末端表现出最大的耐受性。相比之下，在盖和核心结构域中保留功能更多地取决于 AK 的熔点。这些结果表明，家族排列分析确定了进化中选择的主要结构，这些结构的动力学对于酶家族的活性至关重要。这些发现还说明了如何将深度突变扫描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容忍度。
