Determinants of Developability and Evolvability of Synthetic Miniproteins as Ligand Scaffolds.
Determinants of Developability and Evolvability of Synthetic Miniproteins as Ligand Scaffolds.
复制标题
合成微蛋白作为配体支架的可开发性和进化性的决定因素。
DOI：
10.1016/j.jmb.2023.168339
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发表时间：
2023
影响因子：
5.6
通讯作者：
Hackel,BenjaminJ
中科院分区：
文献类型：
--
作者：
McConnell,Adam;Batten,SunLi;Hackel,BenjaminJ
结合配体增强了分子治疗和诊断的能力。尽管有一系列的蛋白质支架被设计用于结合，但驱动可发展性和进化性的生物物理因素尚未完全了解。特别是，在小的单结构域蛋白质的背景下，在保持生物物理完整性的同时，工程新功能受到结构框架和进化的结合位点整合的挑战。迷你蛋白挑战了我们蛋白质工程能力的极限，并在生理靶向、多功能构建的模块化和独特的结合模式方面具有优势。在这里，我们评估了超稳定的合成微蛋白的能力，最初设计用于折叠，作为结合支架的功能。我们合成了45个组合库，有109个变体，系统地在两种拓扑结构中变化，每个拓扑结构有五个起始框架和四个或五个不同的，结构不同的范式，以阐明它们对可进化性和可发展性的影响。我们利用酵母对四个靶标的显示结合选择来评估进化能力。高通量测定-通过酵母显示稳定性和通过分裂gfp线可溶性表达。大肠杆菌-测定显影性。全面的、强大的数据集展示了蛋白质拓扑结构、亲代框架、伞形结构和位置如何影响支架性能。一个高度稳定的框架和局部多样性不足以形成一个有效的支架，但是在合成的微蛋白中设计这些元素的几种设计仅仅是为了稳定，从而产生具有有效的可进化性和可发展性的支架库。工程变体折叠良好，热稳定，结合目标具有个位数纳米摩尔亲和力。因此，超稳定的合成微蛋白可以作为可开发、可进化的微支架的前体，具有独特的生理运输、模块化和结合模式的潜力。
Binding ligands empower molecular therapeutics and diagnostics. Despite an array of protein scaffolds engineered for binding, the biophysical elements that drive developability and evolvability are not fully understood. In particular, engineering novel function while maintaining biophysical integrity within the context of small, single-domain proteins is challenged by integration of the structural framework and the evolved binding site. Miniproteins present a challenge to our limits of protein engineering capability and provide advantages in physiological targeting, modularity for multi-functional constructs, and unique binding modes. Herein, we evaluate the ability of hyperstable synthetic miniproteins, originally designed for foldedness, to function as binding scaffolds. We synthesized 45 combinatorial libraries, with 109variants, systematically varied across two topologies, each with five starting frameworks and four or five diverse, structurally distinct paratopes, to elucidate their impact on evolvability and developability. We evaluated evolvability with yeast display binding selections against four targets. High-throughput assays –stability via yeast display and soluble expression via split-GFP inE. coli– measured developability. The comprehensive, robust dataset demonstrates how protein topology, parental framework, and paratope structure and location all impact scaffold performance. A hyperstable framework and localized diversity are not sufficient for an effective scaffold, but several designs of these elements within synthetic miniproteins designed solely for stability result in scaffold libraries with effective evolvability and developability. Engineered variants were well-folded, thermally stable, and bound target with single-digit nanomolar affinity. Thus, hyperstable synthetic miniproteins can serve as precursors to developable, evolvable mini-scaffolds with unique potential for physiological transport, modularity, and binding modes.