Synthesis of Novel BRET/FRET Protein Probes Containing Light-Emitting Proteins and Fluorescent Nonnatural Amino Acids

• Published in: Bulletin of the Chemical Society of Japan Vol. 85; no. 5; pp. 576 - 583
• Main Authors: Yamaguchi, Atsushi, Hohsaka, Takahiro
• Format: Journal Article
• Language: English
• Published: Tokyo The Chemical Society of Japan 15.05.2012; Chemical Society of Japan
• Subjects: Amino acids; Emission; Fretting; Ligands; Luminescence; Maltose; Proteins; Translations
• ISSN: 0009-2673; 1348-0634
• DOI: 10.1246/bcsj.20110368

Novel light-emitting protein probes were designed and synthesized by incorporating fluorescent nonnatural amino acids and fusing light-emitting proteins. Gaussia luciferase was fused as a BRET donor to the C-terminus of maltose-binding protein, and BODIPY558–aminophenylalanine was incorporated into Tyr210, which is located near the maltose-binding site of maltose-binding protein in response to a four-base codon (CGGG). In addition to the luminescence of luciferase, the double-labeled protein showed the emission of BODIPY558, which was strongly quenched by a neighboring tryptophan in the absence of maltose but was recovered in the presence of maltose. The emission intensity ratio of luciferase and BODIPY558 increased 4.0-fold upon the addition of maltose. Similar ligand-dependent emission changes were observed for a translation reaction mixture expressing the double-labeled protein, indicating that the BRET protein probe was detectable in the presence of excess fluorescent molecules. BODIPY558-containing maltose-binding protein fused with GFP in place of luciferase was also synthesized and showed the FRET and maltose-dependent fluorescence of BODIPY558. Novel BRET/FRET protein probes synthesized by fusing light-emitting proteins and incorporating fluorescent nonnatural amino acids will become valuable tools for the bioimaging and diagnostic detection of specific ligands.