title: A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Utah School of Medicine , 15 North Medical Drive East, Room 4100, Salt Lake City, Utah 84112-5650, United States.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Jacobsen
authors.affiliation: Department of Biochemistry, University of Utah School of Medicine , 15 North Medical Drive East, Room 4100, Salt Lake City, Utah 84112-5650, United States.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Petersen
authors.affiliation: Department of Chemistry & Biochemistry, 8051 Regents Drive, University of Maryland , College Park, Maryland 20742-4454, United States.
authors.forename: Xiang
authors.initials: X
authors.lastname: Ye
authors.affiliation: Centre de Biophysique Moléculaire, CNRS UPR4301 , Rue Charles Sadron, Orléans CEDEX 2 45071, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Galibert
authors.affiliation: Department of Chemistry & Biochemistry, 8051 Regents Drive, University of Maryland , College Park, Maryland 20742-4454, United States.
authors.forename: George H
authors.initials: GH
authors.lastname: Lorimer
authors.affiliation: Centre de Biophysique Moléculaire, CNRS UPR4301 , Rue Charles Sadron, Orléans CEDEX 2 45071, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Aucagne
authors.affiliation: Department of Biochemistry, University of Utah School of Medicine , 15 North Medical Drive East, Room 4100, Salt Lake City, Utah 84112-5650, United States.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Kay
mesh_terms: D000595:Amino Acid Sequence; D060326:Chemistry Techniques, Synthetic; D005449:Fluorenes / Q000737:chemistry; D017510:Protein Folding; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry*; D012269:Ribosomal Proteins / Q000138:chemical synthesis / Q000737:chemistry; D012995:Solubility; D014764:Viral Proteins / Q000737:chemistry
chemical_list: C496789:9-fluorenylmethoxycarbonyl; D005449:Fluorenes; D011506:Proteins; D012269:Ribosomal Proteins; D014764:Viral Proteins; C050985:ribosomal protein L31