Fluorescence resonance energy transfer between unnatural amino acids in a structurally modified dihydrofolate reductase.
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.
Anderson
Raymond D
RD
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.
Zhou
Jia
J
Hecht
Sidney M
SM
D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005453:Fluorescence; D016333:HIV Protease / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011422:Propionates / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry*
C466486:N-dabcyl-1,2-diaminopropionic acid; D010446:Peptide Fragments; D011422:Propionates; D011993:Recombinant Fusion Proteins; C006657:7-azatryptophan; D014364:Tryptophan; D013762:Tetrahydrofolate Dehydrogenase; D016333:HIV Protease