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

Bixun Wang, Kathlynn C. Brown, Michiel Lodder, Charles S. Craik, Sidney M. Hecht

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

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 (Kis) comparable to that of wild-type ecotin. Iodine treatment of ecotin analogue Met84AGly 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 Met84AGly. 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.

Original languageEnglish (US)
Pages (from-to)2805-2813
Number of pages9
JournalBiochemistry
Volume41
Issue number8
DOIs
StatePublished - Feb 26 2002
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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