| Autor: |
Fottner M; Laboratory for Organic Chemistry (LOC), Department of Chemistry and Applied Biosciences (D-CHAB), ETH Zurich, Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland., Heimgärtner J; Laboratory for Organic Chemistry (LOC), Department of Chemistry and Applied Biosciences (D-CHAB), ETH Zurich, Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland., Gantz M; Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, 85748 Garching, Germany., Mühlhofer R; Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, 85748 Garching, Germany., Nast-Kolb T; Center for Protein Assemblies (CPA) and Lehrstuhl für Biophysik (E27), Physics Department, Technical University of Munich, Ernst-Otto-Fischer-Str. 8, 85748 Garching, Germany., Lang K; Laboratory for Organic Chemistry (LOC), Department of Chemistry and Applied Biosciences (D-CHAB), ETH Zurich, Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland.; Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, 85748 Garching, Germany. |
| Abstrakt: |
Asparaginyl endopeptidases (AEPs) have recently been widely utilized for peptide and protein modification. Labeling is however restricted to protein termini, severely limiting flexibility and scope in creating diverse conjugates as needed for therapeutic and diagnostic applications. Here, we use genetic code expansion to site-specifically modify target proteins with an isopeptide-linked glycylglycine moiety that serves as an acceptor nucleophile in AEP-mediated transpeptidation with various probes containing a tripeptidic recognition motif. Our approach allows simple and flexible labeling of recombinant proteins at any internal site and leaves a minimal, entirely peptidic footprint (NGG) in the conjugation product. We show site-specific labeling of diverse target proteins with various biophysical probes, including dual labeling at an internal site and the N-terminus. Furthermore, we harness AEP-mediated transpeptidation for generation of ubiquitin- and ubiquitin-like-modifier conjugates bearing a native isopeptide bond and only one point mutation in the linker region. |
