title: Concatenation of cyan and yellow fluorescent proteins for efficient resonance energy transfer.
abstract: Highly efficient fluorescence resonance energy transfer between cyan(CFP) and yellow fluorescent proteins (YFP), the cyan- and yellow-emitting variants of the Aequorea green fluorescent protein, respectively, was achieved by tightly concatenating the two proteins. After the C-terminus of CFP and the N-terminus of YFP were truncated by 11 and 5 amino acids, respectively, the proteins were fused through a leucine-glutamate dipeptide. The resulting chimeric protein, which we called Cy11.5, exhibited a simple emission spectrum that peaked at 527 nm when the protein was excited at 436 nm. The time-resolved emission of Cy11.5 was measured using a streak camera. After excitation of Cy11.5 with a 400 nm ultrashort pulse, a fast decay of the CFP emission and a concomitant rise of the YFP emission were observed with a lifetime of 66 ps. By contrast, the emission from CFP alone showed a decay component with a lifetime of 2.9 ns. We concluded that in fully folded Cy11.5 molecules, intramolecular FRET occurred with an efficiency of 98%. Importantly, most Cy11.5 molecules were properly folded, and the protein was highly resistant to all of the tested proteases. In living cells, therefore, Cy11.5 behaved as a single fluorescent protein with a broad excitation spectrum. Moreover, Cy11.5 was used as an optical highlighter after photobleaching of YFP. When HeLa cells expressing Cy11.5 were irradiated at 514.5 nm, a 10-fold increase in the 475 nm fluorescence intensity was observed. These features make Cy11.5 useful as an optical highlighter and a new-colored fluorescent protein for multicolor imaging.
authors.affiliation: Laboratory for Cell Function Dynamics, Brain Science Institute, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Shimozono
authors.forename: Haruko
authors.initials: H
authors.lastname: Hosoi
authors.forename: Hideaki
authors.initials: H
authors.lastname: Mizuno
authors.forename: Takashi
authors.initials: T
authors.lastname: Fukano
authors.forename: Tahei
authors.initials: T
authors.lastname: Tahara
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
mesh_terms: D016880:Anisotropy; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D007834:Lasers; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism / Q000494:pharmacology; D038761:Photobleaching; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D010447:Peptide Hydrolases