Protein footprinting by the combined use of reversible and irreversible lysine modifications.
A two-step lysine-modification procedure has been devised to chemically footprint protein surfaces involved in macromolecular interactions. A protein tagged at one particular end, in the free state or in a complex, is first treated lightly with a reversible lysine-modifying reagent. The protein is then unfolded and treated extensively with an irreversible lysine reagent to block those lysines that did not react previously; next, the first lysine modification is reversed, and a lysine-specific 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 lysines. In this procedure, the reversible lysine reagent serves as the chemical footprinting agent, as cleavage of the polypeptide ensues only at the sites of reaction with this reagent. Lysines involved in macromolecular contacts are identified from differences in proteolytic patterns 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.
Hanai
R
R
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138.
Wang
J C
JC
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008239:Lysine; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D014616:Vaccinia virus / Q000201:enzymology
D010455:Peptides; D011506:Proteins; D004247:DNA; D004264:DNA Topoisomerases, Type I; D008239:Lysine