An orange fluorescent protein with a large stokes shift for single-excitation multicolor FCCS and FRET imaging

Daria Shcherbakova, Mark A. Hink, Linda Joosen, Theodorus W J Gadella, Vladislav Verkhusha

Research output: Contribution to journalArticle

112 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)7913-7923
Number of pages11
JournalJournal of the American Chemical Society
Volume134
Issue number18
DOIs
StatePublished - May 9 2012

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Proteins
Imaging techniques
Protons
Lasers
Mutagenesis
Laser excitation
Proton transfer
Energy Transfer
Biosensing Techniques
Photons
Excited states
Biosensors
Isotopes
Energy transfer
Luminance
Calcium
Spectrum Analysis
Color
Fluorescence
Spectroscopy

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

An orange fluorescent protein with a large stokes shift for single-excitation multicolor FCCS and FRET imaging. / Shcherbakova, Daria; Hink, Mark A.; Joosen, Linda; Gadella, Theodorus W J; Verkhusha, Vladislav.

In: Journal of the American Chemical Society, Vol. 134, No. 18, 09.05.2012, p. 7913-7923.

Research output: Contribution to journalArticle

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