Chemically mediated site-specific proteolysis. Alteration of protein-protein interaction

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 (Kis) 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. We have recently described a novel strategy for site- specific cleavage of proteins (1). This technique allows cleavage of the protein amide backbone at a single, pre- determined site with a simple chemical reagent. The strategy involves the use of protein analogues containing the non- native amino acid allylglycine ( A Gly) 1 at a specific site. This amino acid can be attached to suppressor tRNACUA as an activated ester by chemical misacylation (2-9); readthrough of a nonsense (UAG) codon (10-15) at a predetermined site by the use of a suitable protein synthesizing system afforded full-length protein containing A Gly at that site. Subsequent iodine treatment resulted in protein cleavage at the expected site through a presumed iodolactone intermediate. This strategy has been employed successfully to activate a trypsin from its zymogen form (trypsinogen) by chemical removal of the activation peptide, suggesting the compatibility of this technique with the integrity of functional proteins ( 16).