57] Peptidyl-tRNA analogs as affinity labeling agents

Abstract

Publisher Summary Affinity labeling refers to the technique in which a chemically reactive group is attached to a biological molecule so that after formation of a specific reversible complex with its receptor, a unimolecular reaction will occur to couple the receptor and ligand by a covalent bond. This chapter described method that utilizes hydroxysuccinimide esters of carboxylic acids that possess chemically reactive groups separated from the carbonyl group by a “spacer.” The chapter reports a number of reagents that may be utilized to prepare a “survey” series of peptidyl-tRNA analogs containing reactive chemical groups that are capable of forming covalent bonds with most nucleophilic groups of proteins. The chapter also includes methods for the preparation of the affinity labeling agents, controls required for their effective utilization, and suggests criteria to distinguish between nonspecific bimolecular reaction and affinity labeling. Although the chapter focuses on the utilization of tRNA Ile analogs as affinity labeling agents of Ile-tRNA synthetase, the procedures described stress general applicability to all proteins that bind to peptidyl-tRNA.