Methods Enzymol. 2015;556:527-47
- 1Microbiology Department, University of Tennessee. Electronic address: email@example.com.
- 2Chemistry Department, College of Staten Island, City University of New York.
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.