Characterizing and measuring the interactome of N-degrons and N-recognins are critical to the identification and verification of putative N-terminally arginylated native proteins and small-molecule chemicals that structurally and physiologically mimic the N-terminal arginine residue. This chapter focuses on in vitro and in vivo assays to confirm the putative interaction, and measure the binding affinity, between Nt-Arg-carrying natural (or Nt-Arg-mimicking synthetic) ligands and proteasomal or autophagic N-recognins carrying the UBR box or the ZZ domain. These methods, reagents, and conditions are applicable across a wide spectrum of different cell lines, primary cultures, and/or animal tissues, allowing for the qualitative analysis and quantitative measurement of the interaction of arginylated proteins and N-terminal arginine-mimicking chemical compounds to their respective N-recognins.
Keywords: ATE1; Microscale thermophoresis (MST); Nt-arginine-mimicking chemical compound; Nt-arginylation; Pull-down assay.
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.