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 Citations (Scopus)

Abstract

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
Volume124
Issue number33
DOIs
StatePublished - Aug 21 2002
Externally publishedYes

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Tetrahydrofolate Dehydrogenase
Fluorescence Resonance Energy Transfer
Amino acids
Proteins
Amino Acids
Fluorescence
Fusion reactions
Transfer RNA
Oxidoreductases
Quenching
Codon
Messenger RNA
Acids
Light
Substrates
Human immunodeficiency virus 1 p16 protease

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Fluorescence resonance energy transfer between unnatural amino acids in a structurally modified dihydrofolate reductase. / Anderson, Raymond D.; Zhou, Jia; Hecht, Sidney M.

In: Journal of the American Chemical Society, Vol. 124, No. 33, 21.08.2002, p. 9674-9675.

Research output: Contribution to journalArticle

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