Artigo Acesso aberto Revisado por pares

An Orange Fluorescent Protein with a Large Stokes Shift for Single-Excitation Multicolor FCCS and FRET Imaging

2012; American Chemical Society; Volume: 134; Issue: 18 Linguagem: Inglês

10.1021/ja3018972

ISSN

1943-2984

Autores

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

Tópico(s)

Photosynthetic Processes and Mechanisms

Resumo

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.

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