Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158.
Department of Biochemistry, University of Washington, Seattle, WA 98195.
Proc Natl Acad Sci U S A. 2017 Oct 10;114(41):10852-10857. doi: 10.1073/pnas.1710695114. Epub 2017 Sep 25.
The folding of natural proteins typically relies on hydrophobic packing, metal binding, or disulfide bond formation in the protein core. Alternatively, a 3D structure can be defined by incorporating a multivalent cross-linking agent, and this approach has been successfully developed for the selection of bicyclic peptides from large random-sequence libraries. By contrast, there is no general method for the de novo computational design of multicross-linked proteins with predictable and well-defined folds, including ones not found in nature. Here we use Rosetta and Tertiary Motifs (TERMs) to design small proteins that fold around multivalent cross-linkers. The hydrophobic cross-linkers stabilize the fold by macrocyclic restraints, and they also form an integral part of a small apolar core. The designed CovCore proteins were prepared by chemical synthesis, and their structures were determined by solution NMR or X-ray crystallography. These mesosized proteins, lying between conventional proteins and small peptides, are easily accessible either through biosynthetic precursors or chemical synthesis. The unique tertiary structures and ease of synthesis of CovCore proteins indicate that they should provide versatile templates for developing inhibitors of protein-protein interactions.
天然蛋白质的折叠通常依赖于疏水包装、金属结合或蛋白质核心中的二硫键形成。或者,可以通过引入多价交联剂来定义 3D 结构,并且已经成功地开发了这种方法来从大型随机序列文库中选择双环肽。相比之下,目前还没有用于从头计算设计具有可预测和明确定义折叠的多交联蛋白质的通用方法,包括自然界中不存在的折叠。在这里,我们使用 Rosetta 和三级基序 (TERMs) 来设计围绕多价交联剂折叠的小蛋白。疏水交联剂通过大环限制稳定折叠,并且它们也是小非极性核心的一个组成部分。设计的 CovCore 蛋白通过化学合成制备,并通过溶液 NMR 或 X 射线晶体学确定其结构。这些中等大小的蛋白介于传统蛋白和小肽之间,无论是通过生物合成前体还是化学合成都很容易获得。CovCore 蛋白独特的三级结构和易于合成表明,它们应该为开发蛋白质-蛋白质相互作用抑制剂提供多功能模板。