Site-Specific Protein Labeling and Generation of Defined Ubiquitin-Protein Conjugates Using an Asparaginyl Endopeptidase.

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.

Substrate Profiling of Mitochondrial Caseinolytic Protease P via a Site-Specific Photocrosslinking Approach.

Approaches for profiling protease substrates are critical for defining protease functions, but remain challenging tasks. We combine genetic code expansion, photocrosslinking and proteomics to identify substrates of the mitochondrial (mt) human caseinolytic protease P (hClpP). Site-specific incorporation of the diazirine-bearing amino acid DiazK into the inner proteolytic chamber of hClpP, followed by UV-irradiation of cells, allows to covalently trap substrate proteins of hClpP and to substantiate hClpP's major involvement in maintaining overall mt homeostasis.