Systematic conformation-to-phenotype mapping via limited deep sequencing of proteins

• Published in: Molecular cell 2023-06, Vol.83 (11), p.1936-1952.e7
• Main Authors: Serebryany, Eugene, Zhao, Victor Y., Park, Kibum, Bitran, Amir, Trauger, Sunia A., Budnik, Bogdan, Shakhnovich, Eugene I.
• Format: Article
• Language: English
• Subjects: Chaperone; Conformational landscape; Disulfide bond; Disulfide scanning; Disulfides - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; High-Throughput Nucleotide Sequencing; Kinetic trap; Misfolding avoidance; Periplasm; Protein Conformation; Protein Folding; Protein sequencing; Proteotoxic stress
• ISSN: 1097-2765; 1097-4164; 1097-4164
• 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. [Display omitted] •We carried out double-Cys mutational scanning of E. coli periplasmic chaperone HdeA•Cys-specific chemical cleavage enabled deep sequencing of variant libraries by MS/MS•HdeA’s folded state is highly brittle, and a subset of misfolded states is cytotoxic Serebryany et al. develop a methodology for “deep” disulfide scanning of protein conformations. A library of double-Cys mutants at randomized positions in an intrinsically disordered protein in vivo sheds light on the biophysical and phenotypic properties of the diverse backbone conformations the protein can adopt. Protein-level “deep sequencing” identifies the double-Cys variants.