A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy

Nat Biotechnol. 2006 May;24(5):577-81. doi: 10.1038/nbt1207. Epub 2006 Apr 30.


Dual-color fluorescence cross-correlation spectroscopy (FCCS) is a promising technique for quantifying protein-protein interactions. In this technique, two different fluorescent labels are excited and detected simultaneously within a common measurement volume. Difficulties in aligning two laser lines and emission crossover between the two fluorophores, however, make this technique complex. To overcome these limitations, we developed a fluorescent protein with a large Stokes shift. This protein, named Keima, absorbs and emits light maximally at 440 nm and 620 nm, respectively. Combining a monomeric version of Keima with cyan fluorescent protein allowed dual-color FCCS with a single 458-nm laser line and complete separation of the fluorescent protein emissions. This FCCS approach enabled sensitive detection of proteolysis by caspase-3 and the association of calmodulin with calmodulin-dependent enzymes. In addition, Keima and a spectral variant that emits maximally at 570 nm might facilitate simultaneous multicolor imaging with single-wavelength excitation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anthozoa
  • Calmodulin / chemistry
  • Fluorescent Dyes / pharmacology
  • Molecular Sequence Data
  • Mutagenesis
  • Mutation
  • Recombinant Proteins / chemistry
  • Spectrometry, Fluorescence / instrumentation*
  • Spectrometry, Fluorescence / methods*
  • Ultracentrifugation


  • Calmodulin
  • Fluorescent Dyes
  • Recombinant Proteins

Associated data

  • GENBANK/AB209967
  • GENBANK/AB209968
  • GENBANK/AB209969