Project Details
Projekt
Establishing strategies for sortase-catalyzed multi-peptide assemblies to profile T-cell selectivity
Subject Area
Biological and Biomimetic Chemistry
Term
from 2015 to 2019
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 283011643
The bacterial transpeptidase sortase A has become a versatile tool for protein chemistry by catalyzing chemoselective ligation reactions of peptides, proteins and synthetic probes. During catalysis sortase cleaves a so-called sorting motif consisting of the amino acid sequence LPxTG or LPxTA at the threonine residue under concomitant formation of a sortase-bound thioester. The enzyme is deacylated by ligating the thioester to the glycine residue of a second peptide that serves as nucleophile. While sortase A of S. aureus is widely used in biochemistry and chemical biology the assembly of multiple peptides is limited by the fact that each sorting motif and nucleophile is recognized by sortase, thereby leading to fragment shuffling and polymerization as a manifesting side reaction when fragment numbers increase. We plan to address these problems by establishing switchable ligation sites. Sorting motif and/or N-Gly nucleophile will be installed in either active on-states, allowing sortase-mediated ligation, or inactive off-state. Simple chemical conversions will allow switching between states and enable multi-fragment assemblies. These developments will go hand-in-hand with directed evolution of sortase selectivity and activity aimed to (a) engineer sortase mutants specific for ligation site switching approaches as well as (b) to generate new sortases with orthogonal substrate selectivity. We plan to use these new tools for establishing tailor-made probes for immunology, in particular for the investigation of T-cell activation. Antigen peptide tetramers equipped with biophysical probes will be assembled by the methods described above. Avidity effects of the assembled antigen tetramers will be exploited for subsequent orthogonal ligation with MHCII molecules and T-cells. These tools should be useful to study T-cell activation and for tracking rare T-cell populations in infection or autoimmune disease models.
DFG Programme
Priority Programmes
