Флуоресцентные белки красной спектральной области

An imaging of individual molecules, intracellular organelles, live cells and whole model organisms is one of the most important scientific problems in the fields of biochemistry, biotechnology, cell and developmental biology. At present, genetically encoded fluorescent proteins, such as the green fluorescent protein (GFP) and its homologues, including the new red fluorescent proteins are widely used to solve these problems. In this minireview we summarize spectral characteristics, biochemical properties and potential applications of the GFP-like proteins and, particularly, the red fluorescent proteins.Визуализация отдельных молекул, внутриклеточных структур, живых клеток и целых модельных организмов является одной из наиболее важных проблем при проведении исследований в области биохимии, биотехнологии, клеточной биологии и биологии развития. В настоящее время для решения таких научных задач широко используются генетически кодируемые флуоресцентные белки, такие как зелёный флуоресцентный белок (GFP) и ему гомологичные, в том числе и новые красные флуоресцентные белки. В этом миниобзоре суммируются сведения о спектральных характеристиках, биохимических свойствах и перспективах применения GFP-подобных белков и, в особенности, красных флуоресцентных белков

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

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 5-fold larger than that of the brightest red LSSFP and similar to the green-yellow LSS-FPs. LSSmOrange allows numerous multicolor applications using a single excitation wavelength that was not possible before. Using LSSmOrange we developed a 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 the particles labeled with four FPs. LSSmOrange was further applied to simultaneously image two Forster resonance energy transfer pairs, one of which is the commonly used CFP-YFP pair, with a single excitation laser. 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

Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET

SummaryWe have developed the first red fluorescent protein, named rsTagRFP, which possesses reversibly photoswitchable absorbance spectra. Illumination with blue and yellow light switches rsTagRFP into a red fluorescent state (ON state) or nonfluorescent state (OFF state), respectively. The ON and OFF states exhibit absorbance maxima at 567 and 440 nm, respectively. Due to the photoswitchable absorbance, rsTagRFP can be used as an acceptor for a photochromic Förster resonance energy transfer (pcFRET). The photochromic acceptor facilitates determination of a protein-protein interaction by providing an internal control for FRET. Using pcFRET with EYFP as a donor, we observed an interaction between epidermal growth factor receptor and growth factor receptor-binding protein 2 in live cells by detecting the modulation of both the fluorescence intensity and lifetime of the EYFP donor upon the ON-OFF photoswitching of the rsTagRFP acceptor

Флуоресцентные белки красной спектральной области

An imaging of individual molecules, intracellular organelles, live cells and whole model organisms is one of the most important scientific problems in the fields of biochemistry, biotechnology, cell and developmental biology. At present, genetically encoded fluorescent proteins, such as the green fluorescent protein (GFP) and its homologues, including the new red fluorescent proteins are widely used to solve these problems. In this minireview we summarize spectral characteristics, biochemical properties and potential applications of the GFP-like proteins and, particularly, the red fluorescent proteins.Визуализация отдельных молекул, внутриклеточных структур, живых клеток и целых модельных организмов является одной из наиболее важных проблем при проведении исследований в области биохимии, биотехнологии, клеточной биологии и биологии развития. В настоящее время для решения таких научных задач широко используются генетически кодируемые флуоресцентные белки, такие как зелёный флуоресцентный белок (GFP) и ему гомологичные, в том числе и новые красные флуоресцентные белки. В этом миниобзоре суммируются сведения о спектральных характеристиках, биохимических свойствах и перспективах применения GFP-подобных белков и, в особенности, красных флуоресцентных белков

Effect of high pressure and reversed micelles on the fluorescent proteins

Two physico-chemical perturbations were applied to ECFP, EGFP, EYFP and DsRed fluorescent proteins: high hydrostatic pressure and encapsulation in reversed micelles. The observed fluorescence changes were described by two-state model and quantified by thermodynamic formalism. ECFP, EYFP and DsRed exhibited similar reaction volumes under pressure. The changes of the chemical potentials of the chromophore in bis(2-ethylhexyl)sulfosuccinate (AOT) micelles caused apparent chromophore protonation changes resulting in a fluorescence decrease of ECFP and EYFP. In contrast to the remarkable stability of DsRed, the highest sensitivity of EYFP fluorescence under pressure and in micelles is attributed to its chromophore structure. (C) 2003 Elsevier B.V. All rights reserved