A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis

• Published in: Journal of the American Chemical Society Vol. 138; no. 36; pp. 11775 - 11782
• Main Authors: Jacobsen, Michael T, Petersen, Mark E, Ye, Xiang, Galibert, Mathieu, Lorimer, George H, Aucagne, Vincent, Kay, Michael S
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 14-09-2016
• Subjects: Amino Acid Sequence; Chemistry Techniques, Synthetic; Fluorenes - chemistry; Life Sciences; Protein Folding; Proteins - chemical synthesis; Proteins - chemistry; Ribosomal Proteins - chemical synthesis; Ribosomal Proteins - chemistry; Solubility; Viral Proteins - chemistry
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.6b05719

Although native chemical ligation (NCL) and related chemoselective ligation approaches provide an elegant method to stitch together unprotected peptides, the handling and purification of insoluble and aggregation-prone peptides and assembly intermediates create a bottleneck to routinely preparing large proteins by completely synthetic means. In this work, we introduce a new general tool, Fmoc-Ddae-OH, N-Fmoc-1-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-3-[2-(2-aminoethoxy)­ethoxy]-propan-1-ol, a heterobifunctional traceless linker for temporarily attaching highly solubilizing peptide sequences (“helping hands”) onto insoluble peptides. This tool is implemented in three simple and nearly quantitative steps: (i) on-resin incorporation of the linker at a Lys residue ε-amine, (ii) Fmoc-SPPS elongation of a desired solubilizing sequence, and (iii) in-solution removal of the solubilizing sequence using mild aqueous hydrazine to cleave the Ddae linker after NCL-based assembly. Successful introduction and removal of a Lys6 helping hand is first demonstrated in two model systems (Ebola virus C20 peptide and the 70-residue ribosomal protein L31). It is then applied to the challenging chemical synthesis of the 97-residue co-chaperonin GroES, which contains a highly insoluble C-terminal segment that is rescued by a helping hand. Importantly, the Ddae linker can be cleaved in one pot following NCL or desulfurization. The purity, structure, and chaperone activity of synthetic l-GroES were validated with respect to a recombinant control. Additionally, the helping hand enabled synthesis of d-GroES, which was inactive in a heterochiral mixture with recombinant GroEL, providing additional insight into chaperone specificity. Ultimately, this simple, robust, and easy-to-use tool is expected to be broadly applicable for the synthesis of challenging peptides and proteins.