Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
Mol Cell. 2023 Jun 1;83(11):1936-1952.e7. doi: 10.1016/j.molcel.2023.05.006.
Non-native conformations drive protein-misfolding diseases, complicate bioengineering efforts, and fuel molecular evolution. No current experimental technique is well suited for elucidating them and their phenotypic effects. Especially intractable are the transient conformations populated by intrinsically disordered proteins. We describe an approach to systematically discover, stabilize, and purify native and non-native conformations, generated in vitro or in vivo, and directly link conformations to molecular, organismal, or evolutionary phenotypes. This approach involves high-throughput disulfide scanning (HTDS) of the entire protein. To reveal which disulfides trap which chromatographically resolvable conformers, we devised a deep-sequencing method for double-Cys variant libraries of proteins that precisely and simultaneously locates both Cys residues within each polypeptide. HTDS of the abundant E. coli periplasmic chaperone HdeA revealed distinct classes of disordered hydrophobic conformers with variable cytotoxicity depending on where the backbone was cross-linked. HTDS can bridge conformational and phenotypic landscapes for many proteins that function in disulfide-permissive environments.
非天然构象驱动蛋白质错误折叠疾病，使生物工程复杂化，并推动分子进化。目前没有一种实验技术能很好地阐明这些构象及其表型效应。特别是由固有无序蛋白质组成的瞬态构象更是难以捉摸。我们描述了一种系统发现、稳定和纯化天然和非天然构象的方法，这些构象可以在体外或体内产生，并直接将构象与分子、机体或进化表型联系起来。这种方法涉及整个蛋白质的高通量二硫键扫描（HTDS）。为了揭示哪些二硫键捕获哪些色谱可分辨的构象，我们设计了一种用于蛋白质双 Cys 变体文库的深度测序方法，该方法可以精确且同时定位每个多肽中的两个 Cys 残基。对丰富的大肠杆菌周质伴侣蛋白 HdeA 的 HTDS 揭示了不同类别的无序疏水性构象，其细胞毒性取决于骨干交联的位置。HTDS 可以为许多在二硫键允许的环境中发挥作用的蛋白质架起构象和表型景观之间的桥梁。
