A diverse array of fluorescent protein genetic variants has emerged over recent years, offering emission profiles that span almost the entire visible spectrum. Through extensive mutagenesis of the original jellyfish protein, researchers have developed new probes ranging from blue to yellow, which are now among the most widely used in vivo reporter molecules in biological research. Longer-wavelength proteins, emitting in the orange and red regions, have been derived from marine organisms like the anemone Discosoma striata and reef corals belonging to the class Anthozoa. Other species have contributed proteins with cyan, green, yellow, orange, red, and far-red fluorescence. Ongoing development focuses on improving brightness and stability to enhance their overall utility.
Understanding the spectral diversity of these proteins requires exploring the stereochemistry of the fluorophore and how its environment influences fluorescent properties. Beyond jellyfish proteins, red-shifted variants show significant fluorophore variation. While the planar cis configuration is common in orange and red proteins, studies have revealed at least two other motifs: planar trans and non-planar trans. For instance, eqFP611 from Entacmaea quadricolor features a planar trans motif, offering one of the largest Stokes shifts among natural Anthozoan proteins. In contrast, the non-planar trans conformation is seen in non-fluorescent chromoproteins like Rtms5 from Montipora efflorescens. Some newer mFruit proteins even exhibit unusual chromophore architectures that deviate from planar geometry.
Key principles have emerged regarding emission color origins and manipulation. The extent of p-orbital conjugation within the chromophore largely determines the spectral class (e.g., cyan, green, yellow, or red). Local environmental factors—such as charged amino acid residues, hydrogen bonding, and hydrophobic interactions—can shift absorption and emission maxima by up to 20 nm. As research unravels the structure-function relationships of chromophores, engineering finely tuned color variants and expanding the spectral range will become more achievable.
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Blue Fluorescent Proteins
Blue fluorescent proteins (BFPs), emitting between 440–470 nm, were first created by mutating tyrosine at position 66 in the GFP chromophore to histidine (Y66H). This variant absorbs near 380 nm and emits at 448 nm. Early versions were dim, with only 15–20% of GFP’s brightness, due to low quantum yield. Enhanced versions reached 25% of EGFP’s brightness but still suffered from limited photostability.
Developed primarily for FRET experiments and multicolor labeling in the 1990s, BFP’s emission is easily distinguishable from GFP, making it useful for early multicolor imaging. It was also part of the first genetically encoded biosensor. However, BFP requires ultraviolet excitation, which is phototoxic to cells, and faces challenges like autofluorescence, high absorption, and light scattering. These limitations have reduced interest, though some research continues with non-natural amino acids to create blue-shifted artificial variants.
Cyan Fluorescent Proteins
Cyan fluorescent proteins (CFPs), emitting from 470–500 nm, emerged alongside BFP when tyrosine was replaced with tryptophan (Y66W). This single mutation produced a chromophore absorbing at 436 nm and emitting broadly at 485 nm. Refinements like F64L and S65T led to enhanced CFP (ECFP), though brightness remained at 40% of EGFP. ECFP found use as a FRET partner with yellow proteins and for time-lapse imaging due to better photostability than BFP.
ECFP-EYFP FRET pairs have been widely used but often suffer from small dynamic range and dimerization issues. Monomeric variants (mCFP and mYFP) were created by introducing the A206K mutation, improving FRET efficiency. Newer cyan proteins like Cerulean and CyPet offer better performance. Cerulean, with higher extinction coefficient and quantum yield, is twice as bright as ECFP and ideal for FRET with Venus. CyPet, optimized with YPet for FRET, provides six times higher dynamic range than CFP-YFP but expresses poorly at 37°C.
Anthozoan-derived cyan proteins include AmCyan1 from Anemonia majano, which has better photostability than CFP but forms tetramers. Midori-Ishi Cyan (MiCy) from Acropora coral pairs well with mKO for FRET, with high spectral overlap. Monomeric teal fluorescent protein (mTFP1) from Clavularia soft coral offers superior brightness, pH insensitivity, and photostability, making it an excellent FRET donor.
Green Fluorescent Proteins
The original green fluorescent protein (GFP) from Aequorea victoria has bimodal absorption (395/475 nm) and low extinction coefficients, limiting its practicality. The S65T mutation led to enhanced GFP (EGFP), with a single peak at 484 nm, high brightness, and photostability. EGFP became a staple for live-cell imaging, though it has slight pH sensitivity and weak dimerization tendency.
Other green variants include Emerald, with improved folding and brightness, and Sapphire, which has a large Stokes shift (399 nm excitation, 511 nm emission) ideal for FRET with red proteins. aceGFP from Aequorea coerulescens offers brightness similar to EGFP and functions well as a monomer in fusions.
Copepod-derived proteins like CopGFP and TurboGFP are brighter and pH-resistant but can form aggregates. Coral-derived proteins such as Azami Green and ZsGreen are tetrameric, though monomeric versions of Azami Green exist. Renilla GFP from sea pansy is an obligate dimer but expresses well in various organisms.
Green proteins are also valuable in FRAP experiments to study intracellular dynamics, as demonstrated with EGFP fused to endoplasmic reticulum tags.
Yellow Fluorescent Proteins
Yellow fluorescent proteins (YFPs), emitting from 525–555 nm, are highly versatile. The first YFP was created by mutating threonine 203 to tyrosine (T203Y), shifting spectra by 20 nm. Variants like Topaz and EYFP were developed for brightness and maturation efficiency. EYFP is widely used in multicolor imaging and FRET but is sensitive to pH and chloride ions, dimers weakly, and photobleaches easily.
Citrine introduced the Q69M mutation, improving acid stability and photostability. Venus, with F46L, accelerates chromophore maturation and reduces chloride sensitivity but has poor photostability. YPet, optimized with CyPet for FRET, is the brightest YFP and acid-resistant but remains a weak dimer.
Circularly permuted YFPs (cpYFPs) allow fusions at non-terminal sites, expanding their application range. phiYFP from Phialidium jellyfish is naturally similar to engineered Aequorea proteins, with bright yellow emission. Coral-derived mHoneydew and mBanana emit in the yellow range but are dim and pH-sensitive. ZsYellow from Zoanthus has a unique three-ring chromophore but forms tetramers and is only 25% as bright as EGFP.
Orange Fluorescent Proteins
Orange fluorescent proteins (emitting 555–580 nm) include Kusabira Orange from Fungia concinna, which was engineered into a monomeric version (mKO). mKO has excellent photostability, absorbs at 548 nm, and emits at 561 nm, making it ideal for long-term imaging and FRET with cyan proteins. mOrange, derived from mRFP1, is brighter but less photostable. cOFP from Cerianthus anemone is tetrameric and less characterized.
The Sapphire-mOrange FRET pair offers an alternative to CFP-YFP, with well-separated spectra for efficient energy transfer.
Red Fluorescent Proteins
Red fluorescent proteins (RFPs), emitting from 580–630 nm, are valuable for reduced phototoxicity and deeper tissue imaging. DsRed from Discosoma striata was the first major breakthrough, emitting at 583 nm but maturing slowly through a green intermediate and forming tetramers. DsRed2 and DsRed-Express improved maturation rates and reduced aggregation but remained tetrameric.
Monomeric variants like mRFP1 were dim and unstable. Directed evolution produced the "fruit" series, including mStrawberry, mCherry, and tdTomato. mCherry (587/610 nm) is photostable and useful for long-term imaging, while tdTomato is bright but a tandem dimer. eqFP611 from Entacmaea quadricolor emits at 611 nm with a large Stokes shift and reduced oligomerization tendency.
Other RFPs like HcRed1, AsRed2, and JRed form oligomers and have limited brightness. Efforts continue to develop bright, monomeric red proteins with improved photostability.
Far-Red Fluorescent Proteins
Far-red proteins (630–700 nm) include mPlum, emitting at 649 nm with excellent photostability but low brightness, and AQ143 from Actinia equina, which forms tetramers. These proteins are useful for multicolor imaging and FRET with green or yellow donors.
Frequently Asked Questions
What are fluorescent proteins used for?
Fluorescent proteins are genetically encoded tags used to visualize and track cellular processes in real time. They serve as reporters for gene expression, markers for protein localization, and biosensors in FRET assays to monitor molecular interactions.
How do fluorescent proteins get their color?
Color is determined by the chromophore's chemical structure and its environment within the protein. Conjugation extent, hydrogen bonding, and nearby amino acids influence excitation and emission wavelengths, allowing tuning from blue to far-red.
What is FRET and how are fluorescent proteins involved?
FRET (Förster Resonance Energy Transfer) is a technique to measure molecular proximity. It involves a donor protein (e.g., CFP) transferring energy to an acceptor (e.g., YFP) when close. Fluorescent proteins are engineered as FRET pairs to study dynamic processes like calcium signaling or protein interactions.
Why are monomeric fluorescent proteins preferred?
Monomeric proteins avoid oligomerization, which can disrupt natural protein function and cause aggregation. They are ideal for fusion tags, ensuring accurate localization and reducing artifacts in live-cell imaging.
What are the challenges with red fluorescent proteins?
Many red proteins form tetramers, mature slowly, and have intermediate green states. While monomeric variants like mCherry exist, achieving high brightness, fast maturation, and good photostability remains a focus of ongoing research.
Can fluorescent proteins be used in multicolor imaging?
Yes, proteins with distinct emission spectra enable simultaneous tracking of multiple targets. For example, cyan, green, yellow, orange, red, and far-red variants can be combined to study complex cellular events, though careful filter selection is needed to minimize bleed-through.
Conclusion
The development of fluorescent proteins continues to advance, with efforts focused on refining blue to yellow variants from Aequorea victoria and creating monomeric orange to far-red proteins. Progress has yielded improved monomers for cyan, green, and yellow regions, plus promising red candidates. Innovations like unnatural amino acids and circular permutation will further expand the palette. These proteins not only serve as powerful biological tools but also reflect natural photo-protection mechanisms in marine organisms. Their diverse optical properties ensure a growing role in research, enabling new insights into cellular dynamics and molecular interactions.