DETECTING DISORDERED REGIONS IN PROTEINS BY LIMITED PROTEOLYSIS

The limited proteolysis technique can be used to analyse protein structure and dynamics and, in particular, to identify disordered sites or regions within otherwise folded globular proteins. The approach relies on the fact that the proteolysis of a polypeptide substrate requires its binding and adaptation at the protease’s active site and thus enhanced backbone flexibility or local unfolding of the site of proteolytic attack. A striking correlation was found between sites of limited proteolysis and sites of enhanced chain flexibility of the polypeptide chain, this last evaluated by the crystallographically determined B-factor. It is herewith shown that often limited proteolysis occurs at chain regions characterized by missing electron density, thus indicating that protein disorder occurs at these regions. It is concluded that limited proteolysis is a very useful and reliable experimental technique that can be used to probe protein structure and dynamics and, in particular, to detect sites of disorder in proteins, thus complementing the results that can be obtained by the use of other physicochemical and computational approaches. The peculiar advantages of this simple biochemical technique include the requirement of very minute amounts of protein sample, thus when other experimental techniques cannot be used.