Mapping protein-ligand interactions by hydroxyl-radical protein footprinting.

Abstract

Hydroxyl-radical protein footprinting is a direct method to map protein sites involved in macromolecular interactions. The first step is to radioactively end-label the protein. Using hydroxyl radicals as a peptide backbone cleavage reagent, the protein is then cleaved in the absence and presence of ligand. Cleavage products are separated by high-resolution gel electrophoresis. The digital image of the footprinting gel can be subjected to quantitative analysis to identify changes in the sensitivity of the protein to hydroxyl-radical cleavage. Molecular weight markers are electrophoresed on the same gel and hydroxyl-radical cleavage sites assigned by interpolation between the known cleavage sites of the markers. The results are presented in the form of a difference plot that show regions of the protein that change their susceptibility to cleavage while bound to a ligand.