Fluorescence resonance energy transfer between unnatural amino acids in a structurally modified dihydrofolate reductase

Raymond D. Anderson, Jia Zhou, Sidney M. Hecht

Research output: Contribution to journalArticle

56 Scopus citations


The cleavage of a substrate protein by HIV-1 protease has been monitored in real time by the use of a dihydrofolate reductase fusion protein in which a fluorescence donor and a fluorescence acceptor were introduced into sites flanking the HIV-1 protease cleavage site. The amino acids 7-azatryptophan and dabcyl-1,2-diaminopropionic acid were introduced into specific sites of the DHFR fusion protein in an in vitro protein biosynthesizing system using two misacylated suppressor tRNAs, each of which recognized a specific, unique codon introduced into the mRNA. Excitation of the fluorescence acceptor in the initially expressed protein afforded no light production, consistent with quenching by fluorescence resonance energy transfer. Treatment of the elaborated protein with HIV-1 protease cleaved the protein between the fluorescence donor and acceptor, affording a time-dependent increase in fluorescence that was equal in magnitude to that produced by admixture of a stoichiometric amount of free 7-azatryptophan to the solution containing the intact protein.

Original languageEnglish (US)
Pages (from-to)9674-9675
Number of pages2
JournalJournal of the American Chemical Society
Issue number33
StatePublished - Aug 21 2002
Externally publishedYes


ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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