Relative tolerance of mesostable and thermostable protein homologs to extensive mutation
Relative tolerance of mesostable and thermostable protein homologs to extensive mutation
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DOI：
10.1002/prot.21227
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发表时间：
2007-02-01
影响因子：
2.9
通讯作者：
Hilvert, Donald
中科院分区：
文献类型：
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作者：
Besenmatter, Werner;Kast, Peter;Hilvert, Donald
蛋白质的可进化性、可设计性和可塑性对蛋白质工程师很重要，但难以量化。在这里，我们直接比较同源的AroQ分支酸突变酶从嗜热菌詹氏甲烷球菌和嗜温大肠杆菌的能力，以适应广泛的突变。基于各自的野生型序列，使用疏水性和亲水性残基的二元模式在遗传水平上随机化包含约40%的这些蛋白质的N-末端螺旋。通过分支酸突变酶缺陷型E.大肠杆菌菌株。功能性变体被发现与其中稳态对应物相比，热稳定蛋白质的频率高出10倍。此外，详细的序列分析表明，功能M。詹氏酶变体含有较少数量的保守残基，并且在单个序列位置上耐受较大的变异性。因此，我们的研究结果突出了更大的鲁棒性的热稳定蛋白质相对于氨基酸取代，同时确定特定的网站构建活性酶的重要性。总的来说，他们支持这样的观点，即重新设计项目将受益于使用热稳定的起始结构，即使在非常高的突变负荷下。
Evolvability, designability, and plasticity of a protein are properties that are important to protein engineers, but difficult to quantify. Here, we directly compare homologous AroQ chorismate mutases from the thermophile Methanococcus jannaschii and the mesophile Escherichia coli with respect to their capacity to accommodate extensive mutation. The N-terminal helix comprising about 40% of these proteins was randomized at the genetic level using a binary pattern of hydrophobic and hydrophilic residues based on the respective wildtype sequences. Catalytically active library members were identified by a survival-selection assay in a chorismate mutase-deficient E. coli strain. Functional variants were found similar to 10-times more frequently with the thermostable protein compared to its mesostable counterpart. Moreover, detailed sequence analysis revealed that functional M. jannaschii enzyme variants contained a smaller number of conserved residues and tolerated greater variability at individual sequence positions. Our results thus highlight the greater robustness of the thermostable protein with respect to amino acid substitution, while identifying specific sites important for constructing active enzymes. Overall, they support the notion that redesign projects will benefit from using a thermostable starting structure, even at very high mutational loads.