title: An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging.
abstract: Multicolor imaging based on genetically encoded fluorescent proteins (FPs) is a powerful approach to study several dynamic processes in a live cell. We report a monomeric orange FP with a large Stokes shift (LSS), called LSSmOrange (excitation/emission at 437/572 nm), which fills up an existing spectral gap between the green-yellow and red LSSFPs. Brightness of LSSmOrange is five-fold larger than that of the brightest red LSSFP and similar to the green-yellow LSSFPs. LSSmOrange allows numerous multicolor applications using a single-excitation wavelength that was not possible before. Using LSSmOrange we developed four-color single-laser fluorescence cross-correlation spectroscopy, solely based on FPs. The quadruple cross-correlation combined with photon counting histogram techniques allowed quantitative single-molecule analysis of particles labeled with four FPs. LSSmOrange was further applied to simultaneously image two Förster resonance energy transfer pairs, one of which is the commonly used CFP-YFP pair, with a single-excitation laser line. The combination of LSSmOrange-mKate2 and CFP-YFP biosensors enabled imaging of apoptotic activity and calcium fluctuations in real time. The LSSmOrange mutagenesis, low-temperature, and isotope effect studies revealed a proton relay for the excited-state proton transfer responsible for the LSS phenotype.
authors.affiliation: Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
authors.forename: Daria M
authors.initials: DM
authors.lastname: Shcherbakova
authors.forename: Mark A
authors.initials: MA
authors.lastname: Hink
authors.forename: Linda
authors.initials: L
authors.lastname: Joosen
authors.forename: Theodorus W J
authors.initials: TW
authors.lastname: Gadella
authors.forename: Vladislav V
authors.initials: VV
authors.lastname: Verkhusha
mesh_terms: D002470:Cell Survival; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins