title: Cross-linking strategies to study peptide ligand-receptor interactions.
abstract: Experiments are described that allowed cross-linking of analogs of a 13-amino acid peptide into the binding site of a model G protein-coupled receptor. Syntheses of peptide analogs that were used for photochemical or chemical cross-linking were carried out using solid-phase peptide synthesis. Chemical cross-linking utilized 3,4-dihydroxy-l-phenylalanine-incorporated peptides and subsequent periodate-mediated activation, whereas photochemical cross-linking was mediated by p-benzoyl-l-phenylalanine (Bpa)-labeled peptides and UV-initiated activation. Mass spectrometry was employed to locate the site(s) in the receptor that formed the cross-links to the ligand. We also describe a method called unnatural amino acid replacement that allowed capture of a peptide ligand into the receptor. In this method, the receptor was genetically modified by replacement of a natural amino acid with Bpa. The modified receptor was UV-irradiated to capture the ligand. The approaches described are applicable to other peptide-binding proteins and can reveal the ligand-binding site in atomic detail.
authors: Microbiology Department, University of Tennessee. Electronic address: jbecker@utk.edu.
authors: Jeffrey M
authors: JM
authors: Becker
authors: Chemistry Department, College of Staten Island, City University of New York.
authors: Fred
authors: F
authors: Naider
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001577:Benzophenones / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*; D060327:Solid-Phase Synthesis Techniques; D014466:Ultraviolet Rays
chemical_list: C436958:1,4-dihydrophenylalanine; C488060:4-benzoylphenylalanine; D001577:Benzophenones; D003432:Cross-Linking Reagents; D008024:Ligands; D010455:Peptides; D043562:Receptors, G-Protein-Coupled; D010649:Phenylalanine
keywords: BPA-labeled peptide; DOPA-labeled peptide; G protein-coupled receptor; Peptide synthesis; Unnatural amino acid replacement