Concatenation of cyan and yellow fluorescent proteins for efficient resonance energy transfer

Biochemistry. 2006 May 23;45(20):6267-71. doi: 10.1021/bi060093i.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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


  • Bacterial Proteins
  • Cyan Fluorescent Protein
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • yellow fluorescent protein, Bacteria
  • Green Fluorescent Proteins
  • Peptide Hydrolases