Nucleic acid sequencing technologies have gone through extraordinary advancements in the past several decades, significantly increasing throughput while reducing cost. To create similar advancement in proteomics, numerous approaches are being investigated to advance protein sequencing. One of the promising approaches uses N-terminal amino acid binders (NAABs), also referred to as recognizers, that selectively can identify amino acids at the N-terminus of a peptide. However, there are only a few engineered NAABs currently available that bind to specific amino acids and meet the requirements of a biotechnology reagent. Therefore, additional NAABs need to be identified and engineered to enable confident identification and, ultimately, de novo protein sequencing. To fill this gap, a human protein GID4 was engineered to create a NAAB for N-terminal proline (Nt-Pro). While native GID4 binds Nt-Pro, its binding is weak (µmol/L) and greatly influenced by the identity of residues following the Nt-Pro. Through directed evolution, yeast-surface display, and fluorescence-activated cell sorting, we identified sequence variants of GID4 with increased binding response to Nt-Pro. Moreover, variants with an A252V mutation showed a reduced influence from residues in the second and third positions of the target peptide when binding to Nt-Pro. The workflow outlined here is shown to be a viable strategy for engineering NAABs, even when starting from native Nt-binding proteins whose binding is strongly impacted by the identity of residues following Nt-amino acid.

中文翻译：

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通过定向进化将 GID4 改造为用作 N 端脯氨酸结合剂


核酸测序技术在过去几十年中取得了非凡的进步，在降低成本的同时显著提高了通量。为了在蛋白质组学领域取得类似的进步，人们正在研究许多方法来推进蛋白质测序。其中一种有前途的方法使用 N 末端氨基酸结合剂 （NAAB），也称为识别器，它可以选择性地识别肽 N 端的氨基酸。然而，目前只有少数工程化的 NAAB 可以与特定氨基酸结合并满足生物技术试剂的要求。因此，需要识别和设计更多的 NAAB，以实现可靠的鉴定，并最终实现从头蛋白质测序。为了填补这一空白，人蛋白 GID4 被改造成 N 末端脯氨酸 （Nt-Pro） 的 NAAB。虽然天然 GID4 与 Nt-Pro 结合，但其结合较弱 （μmol/L），并且受 Nt-Pro 后残基特性的极大影响。通过定向进化、酵母表面展示和荧光激活细胞分选，我们鉴定了对 Nt-Pro 结合反应增加的 GID4 序列变异。此外，具有 A252V 突变的变体在与 Nt-Pro 结合时显示靶肽第二和第三位置残基的影响降低。此处概述的工作流程被证明是工程 NAAB 的可行策略，即使从天然 Nt 结合蛋白开始，其结合受到 Nt-氨基酸后残基特性的强烈影响。