Transpeptidase-catalyzed multi-fragment assemblies of peptides and proteins

• Applicant: Professor Dr. Dirk Schwarzer
• Subject Area: Biochemistry ; Biological and Biomimetic Chemistry
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 527537642

Transpeptidases are powerful tools of protein chemistry by enabling chemoenzymatic ligations of synthetic peptides and recombinant proteins. The central aim of this proposal is the development of new methods for chemoenzymatic ligations of multiple peptides and proteins. We have recently reported multi-peptide assemblies by sortase-mediated ligation (SML) using switchable ligation sites. The transpeptidase sortase A recognizes an LPxTG sorting motif, cleaves the sequence at the threonine residue and ligates the enzyme-bound intermediate to a peptide with N-terminal glycine nucleophile. We showed that the leucine residue can be replaced by a disulfide-protected cysteine allowing SML. Upon disulfide reduction the sorting motif is inactivated in the ligation product. When combined with a photo-caged nucleophile multiple peptides can be ligated by SML. Here we plan to establish switchable ligation sites on protein level by introducing activating disulfides into sorting motifs of recombinant proteins together with protease-activatable nucleophiles. The utility of this approach should be demonstrated by assembling a reporter for inhibitors of acetyllysine binding modules. In a second project, we plan to establish alternative switch sites in the sorting motif focusing on the proline residue. This residue is known to introduce a kink into the conformation of the sorting motif which is essential for sortase binding. By introducing amino acids with acid- or photo-labile backbone modifications, we plan to maintain the kinked conformation during SML and switch to an extended inactive conformation afterwards by acid treatment or UV irradiation. In the last project, we plan to combine sortase and plant transpeptidase butelase 1 in an orthogonal three-fragment ligation scheme by enabling efficient recombinant expression of butelase. Taking the ER localization of the enzyme into account, we plan to evaluate and optimize butelase expression in mammalian Expi293 cells and Leishmania tarentolae, both of which have been optimized for ER targeting of recombinant proteins. A one-pot ligation of the reporter construct for acetyllysine binding module inhibitors should be established, demonstrating utility of this orthogonal ligation scheme.

• DFG Programme: Research Grants