RRC ID 50106
Author Shimozono S, Hosoi H, Mizuno H, Fukano T, Tahara T, Miyawaki A.
Title Concatenation of cyan and yellow fluorescent proteins for efficient resonance energy transfer.
Journal Biochemistry
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.
Volume 45(20)
Pages 6267-71
Published 2006-5-23
DOI 10.1021/bi060093i
PMID 16700538
MeSH Anisotropy Bacterial Proteins / chemistry* Bacterial Proteins / metabolism Fluorescence Resonance Energy Transfer / methods* Green Fluorescent Proteins / chemistry* Green Fluorescent Proteins / metabolism HeLa Cells Humans Kinetics Lasers Luminescent Proteins / chemistry* Luminescent Proteins / metabolism Peptide Hydrolases / metabolism Peptide Hydrolases / pharmacology Photobleaching Recombinant Fusion Proteins / genetics Recombinant Fusion Proteins / metabolism Transfection
IF 2.997
Resource
DNA material Cy11.5/pRSETB (RDB15706).