Engineering at the amino acid level is key to enhancing the properties of existing proteins in a desired manner. So far, protein engineering has been dominated by genetic approaches, which have been extremely powerful but only allow for minimal variations beyond the canonical amino acids. Chemical peptide synthesis allows the unrestricted incorporation of a vast set of unnatural amino acids with much broader functionalities, including the incorporation of post-translational modifications or labels. Here we demonstrate the potential of chemical synthesis to generate proteins in a specific conformation, which would have been unattainable by recombinant protein expression. We use recently established rapid automated flow peptide synthesis combined with solid-phase late-stage modifications to rapidly generate a set of FK506-binding protein 51 constructs bearing defined intramolecular lactam bridges. This trapped an otherwise rarely populated transient pocket─as confirmed by crystal structures─which led to an up to 39-fold improved binding affinity for conformation-selective ligands and represents a unique system for the development of ligands for this rare conformation. Overall, our results show how rapid automated flow peptide synthesis can be applied to precision protein engineering.

中文翻译：

---

自动化流式肽合成使具有稳定瞬时结合袋的蛋白质工程成为可能

氨基酸水平的工程是以所需方式增强现有蛋白质特性的关键。到目前为止，蛋白质工程一直以遗传方法为主，这种方法非常强大，但只允许超出规范氨基酸的最小变化。化学肽合成允许不受限制地掺入大量具有更广泛功能的非天然氨基酸，包括掺入翻译后修饰或标签。在这里，我们展示了化学合成产生特定构象蛋白质的潜力，这是重组蛋白质表达无法实现的。我们使用最近建立的快速自动化流程肽合成技术与固相后期修饰相结合，快速生成一组带有确定的分子内内酰胺桥的 FK506 结合蛋白 51 构建体。这捕获了一个很少填充的瞬时口袋（晶体结构证实了这一点），从而使构象选择性配体的结合亲和力提高了 39 倍，并代表了开发这种罕见构象配体的独特系统。总的来说，我们的结果表明快速自动化流程肽合成如何应用于精密蛋白质工程。