Limited Proteolysis of Protein-Nucleic Acid Complexes

Limited Proteolysis is a useful structural probe for investigating the globular nature of proteins by preferentially digesting the more accessible regions often found between domains. Generally, proteases require a small region of polypeptide chain possessing conformational flexibility for accommodation in the active site (1 ). The regions of a protein possessing conformational flexibility are often found between tightly folded domains and are, therefore, preferential sites for proteolysis. In practice, limited proteolysis is achieved by dilution of the enzyme sufficiently so that it will only digest the most accessible regions leaving the domains intact. Digestion of protein-nucleic acid is often advantageous in that the DNA may provide steric protection of the DNAbinding domain not afforded by the free protein. The generation of domains by limited proteolysis relies directly on the tertiary structure of the protein under investigation and provides much firmer evidence for their existence than that provided by sequence homology.