title: Chemically mediated site-specific proteolysis. Alteration of protein-protein interaction.
abstract: The design and synthesis of a novel iodine-labile serine protease inhibitor was realized by the use of an ecotin analogue containing allylglycine at position 84 in lieu of methionine. Allylglycine-containing ecotins were synthesized by in vitro translation of the ecotin gene containing an engineered nonsense codon (TAG) at the positions of interest. A misacylated suppressor tRNA activated with the unnatural amino acid allylglycine was employed for the suppression of the nonsense codons in a cell-free protein biosynthesizing system, permitting the elaboration of ecotin analogues containing allyglycine at the desired sites. The derived ecotin analogues were capable of inhibiting bovine trypsin with inhibitory constants (K(i)s) comparable to that of wild-type ecotin. Iodine treatment of ecotin analogue Met84(A)Gly resulted in the deactivation of ecotin, caused by peptide backbone cleavage at its P1 reactive site. Upon iodine treatment, active trypsin could be released from the protein complex with ecotin analogue Met84(A)Gly. This constitutes a novel strategy for modulation of serine protease activity and more generally for alteration of protein-protein interaction by a simple chemical reagent.
authors.affiliation: Departments of Chemistry and Biology, University of Virginia, Charlottesville, Virginia 22901, USA.
authors.forename: Bixun
authors.initials: B
authors.lastname: Wang
authors.forename: Kathlynn C
authors.initials: KC
authors.lastname: Brown
authors.forename: Michiel
authors.initials: M
authors.lastname: Lodder
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
authors.forename: Sidney M
authors.initials: SM
authors.lastname: Hecht
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D033903:Periplasmic Proteins; D010957:Plasmids; D011485:Protein Binding; D015842:Serine Proteinase Inhibitors / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D015842:Serine Proteinase Inhibitors