A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy.
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.
Kogure
Takako
T
Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan.
Karasawa
Satoshi
S
Araki
Toshio
T
Saito
Kenta
K
Kinjo
Masataka
M
Miyawaki
Atsushi
A
D000818:Animals; D037421:Anthozoa; D002147:Calmodulin / Q000737:chemistry; D005456:Fluorescent Dyes / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D014461:Ultracentrifugation
D002147:Calmodulin; D005456:Fluorescent Dyes; D011994:Recombinant Proteins