R. Hanai et Jc. Wang, PROTEIN FOOTPRINTING BY THE COMBINED USE OF REVERSIBLE AND IRREVERSIBLE LYSINE MODIFICATIONS, Proceedings of the National Academy of Sciences of the United Statesof America, 91(25), 1994, pp. 11904-11908
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.