FLUORESCENCE-BASED PROTEIN FOOTPRINTING USING HISTIDINE-TAGGED PROTEIN

We describe a procedure for protein footprinting to identify the regio n(s) of a protein that interacts with a ligand. The method utilized th e affinity of a stretch of histidine residues cloned into the protein to metal-chelated resin. After limited protease digestion, the histidi ne-tagged end fragments were separated by the resin and labeled with a fluorescein derivative. Resolving the labeled digestion products on a denaturing polyacrylamide gel and visualizing the peptides using a Fl uorImager(TM) provided a way to identify the protease target sites tha t were protected from digestion because of interaction with DNA. The p rotection experiments would be applicable not only to detect direct co ntact sites but also sites allosterically altered by ligand binding.