Affinity isolation and biochemical characterization of N-degron ligands using the N-recognin, ClpS.

The N-degron pathways are a set of proteolytic systems that relate the half-life of a protein to its N-terminal (Nt) residue. In Escherichia coli the principal N-degron pathway is known as the Leu/N-degron pathway. Proteins degraded by this pathway contain an Nt degradation signal (N-degron) composed of an Nt primary destabilizing (N) residue (Leu, Phe, Trp or Tyr). All Leu/N-degron substrates are recognized by the adaptor protein, ClpS and delivered to the ClpAP protease for degradation. Although many components of the pathway are well defined, the physiological role of this pathway remains poorly understood. To address this gap in knowledge we developed a biospecific affinity chromatography technique to isolate physiological substrates of the Leu/N-degron pathway. In this chapter we describe the use of peptide arrays to determine the binding specificity of ClpS. We demonstrate how the information obtained from the peptide array, when coupled with ClpS affinity chromatography, can be used to specifically elute physiological Leu/N-degron ligands from a bacterial lysate. These techniques are illustrated using E. coli ClpS (EcClpS), but both are broadly suitable for application to related N-recognins and systems, not only for the determination of N-recognin specificity, but also for the identification of natural Leu/N-degron ligands from various bacterial and plant species that contain ClpS homologs.