MAPPING PROTEIN DOMAINS INVOLVED IN MACROMOLECULAR INTERACTIONS - A NOVEL PROTEIN FOOTPRINTING APPROACH

A novel direct approach, analogous to DNA footprinting, for mapping pr otein domains involved in macromolecular interactions is presented in this paper and applied to cAMP receptor protein (CRP) interactions wit h the allosteric ligand (cAMP) and DNA. In this approach, a protein-ma cromolecule complex is subjected to a nonspecific cleavage by Fe-EDTA. The cleavage products are resolved by SDS-PAGE and transferred to a P VDF membrane. Transferred polypeptides are visualized by immunostainin g with antibodies specific to the N-terminal peptide of the protein. T he mobility of the bands visualized in such a way is directly proporti onal to the distance of the cleavage sites from the N-terminus, and th us the positions of the sites protected from cleavage by a bound macro molecule can be determined. Thus, protein domains involved in macromol ecular interactions can be mapped. In the case of CRP, the cleavage co nditions were established which resulted in, on the average, less than one cleavage event/protein molecule and which preserved satisfactory levels of protein and DNA activity. When applied to CRP-DNA interactio ns, the protein footprinting approach correctly identified domains of CRP that were known to be involved in the recognition of DNA. The obta ined results showed also that the binding of CRP to the DNA binding si te perturbed the region of CRP involved in intersubunit interactions. An allosteric ligand (cAMP) appeared to perturb the same region of CRP . This stresses out the importance of intersubunit interactions in cAM P modulation of protein DNA binding affinity. The protein footprinting methodology presented in this paper should be broadly generalizable t o any protein-macromolecule system.