title: Peptide Arrays for Binding Studies of E3 Ubiquitin Ligases.
abstract: The automated SPOT (synthetic peptide arrays on membrane support technique) synthesis technology has entrenched as a rapid and robust method to generate peptide libraries on cellulose membrane supports. The synthesis method is based on conventional Fmoc chemistry building up peptides with free N-terminal amino acids starting at their cellulose-coupled C-termini. Several hundreds of peptide sequences can be assembled with this technique on one membrane comprising a strong binding potential due to high local peptide concentrations. Peptide orientation on SPOT membranes qualifies this array type for assaying substrate specificities of N-recognins, the recognition elements of the N-end rule pathway of targeted protein degradation (NERD). Pioneer studies described binding capability of mammalian and yeast enzymes depending on a peptide's N-terminus. SPOT arrays have been successfully used to describe substrate specificity of N-recognins which are the recognition elements of the N-end rule pathway of targeted protein degradation (NERD). Here, we describe the implementation of SPOT binding assays with focus on the identification of N-recognin substrates, applicable also for plant NERD enzymes.
authors.affiliation: Leibniz Institute of Plant Biochemistry (IPB), Halle (Saale), Germany.|ScienceCampus Halle - Plant-Based Bioeconomy, Halle (Saale), Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Klecker
authors.affiliation: Leibniz Institute of Plant Biochemistry (IPB), Halle (Saale), Germany. nico.dissmeyer@ipb-halle.de.|ScienceCampus Halle - Plant-Based Bioeconomy, Halle (Saale), Germany. nico.dissmeyer@ipb-halle.de.
authors.forename: Nico
authors.initials: N
authors.lastname: Dissmeyer
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D002482:Cellulose / Q000737:chemistry; D008967:Molecular Biology / Q000379:methods*; D009363:Neoplasm Proteins / Q000737:chemistry; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D013379:Substrate Specificity; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D009363:Neoplasm Proteins; D019151:Peptide Library; D010455:Peptides; C021081:recognins; D002482:Cellulose; D044767:Ubiquitin-Protein Ligases
keywords: N-end rule; Peptide library; Protein-protein interaction; ResPep SL; SPOT assay; Substrate screen; Ubiquitin ligase