PROTEIN FOOTPRINTING BY THE COMBINED USE OF REVERSIBLE AND IRREVERSIBLE LYSINE MODIFICATIONS

A two-step lysine-modification procedure has been devised to chemicall y footprint protein surfaces involved in macromolecular interactions. A protein tagged at one particular end, in the free state or in a comp lex, is first treated lightly with a reversible lysine-modifying reage nt, The protein is then unfolded and treated extensively with an irrev ersible lysine reagent to block those lysines that did not react previ ously; next, the first lysine modification is reversed, and a lysine-s pecific endoproteinase is used to cleave the tagged polypeptide at the deblocked lysines. Separation of the proteolytic products by size and identification of the tagged fragments map the positions of these lys ines. In this procedure, the reversible lysine reagent serves as the c hemical footprinting agent, as cleavage of the polypeptide ensues only at the sites of reaction with this reagent. Lysines involved in macro molecular contacts are identified from differences in proteolytic patt erns of the tagged protein when the first lysine modification is done with the protein in the free form and in a complex. Application of the method to vaccinia virus topoisomerase identifies a number of lysines that are involved in its binding to DNA.