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A Combinatorial Histidine Scanning Library Approach to Engineer Highly pH-Dependent Protein Switches

机译:组合组氨酸扫描库方法设计高度依赖pH的蛋白质开关

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摘要

There is growing interest in the development of protein switches, which are proteins whose function, such as binding a target molecule, can be modulated through environmental triggers. Efforts to engineer highly pH sensitive protein–protein interactions typically rely on the rational introduction of ionizable groups in the protein interface. Such experiments are typically time intensive and often sacrifice the protein's affinity at the permissive pH. The underlying thermodynamics of proton-linkage dictate that the presence of multiple ionizable groups, which undergo a pKa change on protein binding, are necessary to result in highly pH-dependent binding. To test this hypothesis, a novel combinatorial histidine library was developed where every possible combination of histidine and wild-type residue is sampled throughout the interface of a model anti-RNase A single domain VHH antibody. Antibodies were coselected for high-affinity binding and pH-sensitivity using an in vitro, dual-function selection strategy. The resulting antibodies retained near wild-type affinity yet became highly sensitive to small decreases in pH, drastically decreasing their binding affinity, due to the incorporation of multiple histidine groups. Several trends were observed, such as histidine “hot-spots,” which will help enhance the development of pH switch proteins as well as increase our understanding of the role of ionizable residues in protein interfaces. Overall, the combinatorial approach is rapid, general, and robust and should be capable of producing highly pH-sensitive protein affinity reagents for a number of different applications.
机译:人们对蛋白质开关的开发越来越感兴趣,蛋白质开关是可以通过环境触发来调节其功能(例如结合靶分子)的蛋白质。进行高度pH敏感的蛋白质与蛋白质相互作用的工程通常依赖于在蛋白质界面中合理引入可电离基团。这样的实验通常是费时的,并且经常在允许的pH值下牺牲蛋白质的亲和力。质子键的潜在热力学表明,存在多个可电离基团(它们在蛋白质结合时发生pKa改变)是导致高度pH依赖性结合所必需的。为了检验该假设,开发了新的组合组氨酸文库,其中在模型抗RNase A单结构域VHH抗体的整个界面上取样组氨酸和野生型残基的每种可能组合。使用体外双重功能选择策略,针对高亲和力结合和pH敏感性共选择抗体。由于并入了多个组氨酸基团,保留的抗体接近野生型亲和力,但对pH的小幅下降变得高度敏感,从而大大降低了它们的结合亲和力。观察到了几种趋势,例如组氨酸“热点”,这将有助于增强pH开关蛋白的发展,并加深我们对可电离残基在蛋白界面中的作用的理解。总体而言,组合方法是快速,通用和可靠的,并且应该能够为许多不同的应用生产高度pH敏感的蛋白亲和试剂。

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