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Fluorescent Proteins

Fluorescent Protein Technology

It took over thirty years, and the advent of recombinant DNA as well as vastly improved molecular biological approaches to see the pioneering work of Osamu Shimomura developed into a useful tool for live-cell imaging by Doug Prasher and Martin Chalfie. Just in the past decade, however, we have witnessed a truly remarkable expansion in the fluorescent protein palette, largely driven by the innovative studies from Roger Tsien's laboratory. Most of the fluorescent proteins that are commonly used today have been modified through mutagenesis to optimize their expression in biological systems. Continued efforts using directed evolution approaches will no doubt improve the spectral characteristics, photostability, maturation time, brightness, acid resistance, and utility of the fluorescent protein tags for cellular imaging.

Review Articles

Introduction to Fluorescent Proteins - The current thrust of fluorescent protein development strategies is centered on fine-tuning the current palette of blue to yellow variants from jellyfish, while simultaneously developing monomeric fluorescent proteins emitting in the orange to far-red regions of the visible light spectrum.

Fluorescent Proteins Derived from Aequorea victoria - We now have jellyfish proteins that span an 80-nanometer portion visible spectrum from deep blue to yellow-green, providing a wide choice of genetically encoded markers for studies in cell biology.

Fluorescent Proteins Derived from Anthozoa - Fluorescent proteins derived from Anthozoa species (corals and anemones), as well as other sources, span the entire visible spectrum and feature a wide range of useful properties.

Optical Highlighter Fluorescent Proteins - The unique optical highlighter properties of fluorescent proteins can allow the investigator to change the color or the emission state, providing unique opportunities to track the dynamic behavior of proteins in living cells and animals.

Practical Considerations for using Fluorescent Proteins - The review in this section provides some general tips for the practical aspects of using and imaging enhanced green fluorescent protein (EGFP), as well as newer members of the color palette.

Interactive Tutorials

Enhanced Green Fluorescent Protein (EGFP) Chromophore Formation - Still the "gold standard" in fluorescent protein technology, the enhanced version of GFP features a chromophore based on a para-hydroxybenzylidene substituted imidazolinone.

DsRed Fluorescent Protein Chromophore Formation - The chromophore of the first reported red fluorescent protein extends conjugation into the polypeptide backbone to generate fluorescence in the longer wavelength regions.

zsYellow Fluorescent Protein Chromophore Formation - The ZsYellow fluorescent protein chromophore features a novel three-ring system and peptide backbone cleavage due to the substitution of lysine for serine as the first amino acid residue in the chromophore tripeptide.

mKusabira Orange Fluorescent Protein Chromophore Formation - The final step in mKO chromophore maturations involves the formation of a novel five-member thiazole ring system when the Cys65 hydroxyl moiety attacks the carbonyl of Phe64 and cyclizes.

mOrange Fluorescent Protein Chromophore Formation - In a manner similar to mKusabira Orange, mOrange chromophore maturation involves the formation of a novel five-member oxazole (rather than a thiazole) ring system.

eqFP611 Chromophore Formation - A planar trans motif is found in the chromophore of the red fluorescent protein eqFP611, isolated from a sea anemone, and displays one of the largest Stokes shifts and red-shifted emission wavelength profiles of any naturally occurring fluorescent protein.

HcRed Fluorescent Protein Chromophore Formation - Although HcRed shares only approximately 21 percent amino acid sequence homology with GFP, enough critical amino acid motifs are conserved to form a very stable three-dimensional beta-barrel structure.

Kaede Fluorescent Protein Chromophore Formation - Upon illumination of the green species with ultraviolet light, the Kaede chromophore undergoes polypeptide chain cleavage between His62 and Phe61 to generate red fluorescence.

Kindling Fluorescent Protein (KFP1) Chromophore Formation - Investigations into the mechanism of kindling fluorescent protein photoswitching suggest that a cis-trans isomerization of the hydroxybenzilidine chromophore moiety is a key event in the switching process.

PA-GFP Chromophore Photoactivation - By replacing the threonine at position 203 with a histidine residue in wild-type GFP, researchers produced a variant having negligible absorbance in the region between 450 and 550 nanometers, thus dramatically enhancing contrast.

Dronpa Fluorescent Protein Chromophore Photoswitching - The most prominent and well-studied photoswitchable fluorescent protein is named Dronpa (named after a fusion of the Ninja term for vanishing and photoactivation), which is a monomeric variant derived from a stony coral tetramer.

Photoconversion of Kaede/Eos Highlighters - Unlike photoactivatable fluorescent proteins, Kaede and Eos are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions for optical highlighting.

Excited-State Proton Transfer - When excited with ultraviolet light, the tyrosine residue in the neutral chromophore of wild-type GFP becomes a strong acid and transfers a proton through a novel hydrogen bond network in a process known as excited-state proton transfer.

Reference Library

Fluorescent Protein Review Articles - The growing class of fluorescent proteins useful for detecting events in living cells and animals has almost single-handedly launched and fueled a new era in biology and medicine. These powerful research tools have provided investigators with a mechanism of fusing a genetically encoded optical probe to a practically unlimited variety of protein targets in order to examine living systems using fluorescence microscopy and related technology. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of fluorescent protein technology.

Optical Highlighter Fluorescent Protein Original References - Optical highlighter fluorescent proteins, which include the photoactivatable GFP (PA-GFP), the green-to-red photoconverter Kaede, and the photoswitchable Dronpa, allow direct and controlled activation of distinct molecular pools of the fluorescent proteins within the cell. Listed in this section are key references to many of the original articles describing the discovery and properties of optical highlighters.

Photoactivation and Photoconversion - The ability to selectively initiate or alter fluorescence emission profiles in fluorescent proteins has resulted in the creation of a new class of probes for exploring protein behavior and dynamics in living cells. As the fluorescence intensity or spectral alterations of highlighters generally occur only after photon-mediated conversion, newly synthesized non-photoactivated protein pools remain unobserved and do not complicate experimental results. This section provides sources for selected review articles and original research reports on optical highlighter fluorescent proteins.

Fluorescent Protein FRET Biosensors - Aside from their utility as fusion partners to report on protein localization in multiple colors, fluorescent proteins have also been cleverly used to create highly specific biosensors to monitor a wide spectrum of physiological processes, including pH fluctuations, calcium wave induction, cyclic nucleotide messenger effects, membrane potential differences, signaling, phosphorylation, redox reactions, and apoptosis.

Aequorea victoria (Jellyfish) Fluorescent Proteins - Explore the early research reports involved with elucidating the structure and function of luminescent and fluorescent proteins derived from the Pacific jellyfish, Aequorea victoria. Papers by Osamu Shimomura, Martin Chalfie, and Roger Tsien described research that eventually resulted in the 2008 Nobel Prize in Chemistry being awarded to these investigators.

Anthozoa (coral) Fluorescent Proteins - The search for a red-emitting fluorescent protein with performance attributes similar to those of the enhanced green fluorescent protein (EGFP) from the Aequorea victoria jellyfish (in effect, brightness, photostability, and utility in fusions) has been seen as a critical avenue to providing an important tool for multicolor imaging and in generating new fluorescence resonance energy transfer (FRET) biosensors with spectral profiles in the longer wavelengths.

Fluorescent Protein Engineering - As a class, fluorescent proteins have been subjected to more extensive protein engineering and artificial directed evolution than almost any other category of protein. Such a concentrated effort on these probes is due to the fact that fluorescent proteins are extremely popular tools in the biological sciences and improved variants can provide huge benefits to researchers.