Fluorescence resonance energy transfer as a probe of peptide cyclization catalyzed by nonribosomal thioesterase domains.
Macrocyclization of synthetic peptides by thioesterase (TE) domains excised from nonribosomal peptide synthetases (NRPS) has been limited to peptides that contain TE-specific recognition elements. To alter substrate specificity of these enzymes by evolution efforts, macrocyclization has to be detected under high-throughput conditions. Here we describe a method to selectively detect cyclic peptides by fluorescence resonance energy transfer (FRET). Using this method, picomolar detection limits were easily realized, providing novel entry for kinetic studies of catalyzed macrocyclization. Application of this method also provides an ideal tool to track TE-mediated peptide cyclization in real time. The general utility of FRET-assisted detection of cyclopeptides was demonstrated for two cyclases, namely tyrocidine (Tyc) TE and calcium-dependent antibiotic (CDA) TE. For the latter cyclase, this approach was combined with site-directed affinity labeling, opening the possibility for high-throughput enzymatic screening.
Grünewald
Jan
J
Philips-Universität Marburg, Fachbereich Chemie/Biochemie, Hans-Meerweinstrasse, 35043 Marburg, Germany
Kopp
Florian
F
Mahlert
Christoph
C
Linne
Uwe
U
Sieber
Stephan A
SA
Marahiel
Mohamed A
MA
D000345:Affinity Labels; D002384:Catalysis; D003500:Cyclization; D004950:Esterases / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007700:Kinetics; D046908:Peptide Biosynthesis, Nucleic Acid-Independent; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism; D010456:Peptides, Cyclic / Q000032:analysis* / Q000138:chemical synthesis; D017434:Protein Structure, Tertiary; D013869:Thiolester Hydrolases / Q000378:metabolism*; D014440:Tyrocidine / Q000378:metabolism
D000345:Affinity Labels; D010456:Peptides, Cyclic; D014440:Tyrocidine; D004950:Esterases; C475899:tyrocidine thioesterase; D013869:Thiolester Hydrolases; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase