title: A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy.
abstract: 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.
authors.affiliation: Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan.
authors.forename: Takako
authors.initials: T
authors.lastname: Kogure
authors.forename: Satoshi
authors.initials: S
authors.lastname: Karasawa
authors.forename: Toshio
authors.initials: T
authors.lastname: Araki
authors.forename: Kenta
authors.initials: K
authors.lastname: Saito
authors.forename: Masataka
authors.initials: M
authors.lastname: Kinjo
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
mesh_terms: 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
chemical_list: D002147:Calmodulin; D005456:Fluorescent Dyes; D011994:Recombinant Proteins