Chemical and Enzymatic Approaches to C-Terminal Cyclic Imide Formation.

The C-terminal cyclic imide modification is recognized as a degron by the E3 ligase adapter cereblon (CRBN). Reliable methods to generate and measure this modification on CRBN substrates are essential for studying its biological function. In this chapter, we describe two complementary approaches for producing C-terminal cyclic imide-modified peptides and proteins: sortase-mediated transpeptidation and enzymatic conversion by protein carboxymethyltransferase (PCMT1). Additionally, we present analytical strategies, including a time-resolved Förster resonance energy transfer (TR-FRET) assay for measuring binding of substrates to CRBN in vitro and mass spectrometry methods for confirming cyclic imide formation. Together, these protocols provide practical methods for investigating the chemistry and biology of C-terminal cyclic imides.