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.

中文翻译：

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从头开始设计具有独特三级结构的共价约束介孔蛋白支架

天然蛋白质的折叠通常依赖于蛋白质核心中的疏水堆积，金属结合或二硫键形成。可替代地，可以通过掺入多价交联剂来定义3D结构，并且已经成功开发了该方法用于从大型随机序列文库中选择双环肽。相比之下，目前尚无通用的方法可以重新设计具有可预见的和明确定义的折叠（包括自然界中未发现的折叠）的多交联蛋白。在这里，我们使用Rosetta和叔基序（TERM）设计围绕多价交联剂折叠的小蛋白质。疏水性交联剂通过大环限制稳定了折叠，并且它们还形成了小的非极性核的组成部分。通过化学合成制备设计的CovCore蛋白，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质位于常规蛋白质和小肽之间，可通过生物合成前体或化学合成轻松获得。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与蛋白相互作用的抑制剂提供通用的模板。