A Brief History of Fluorescent-Protein Research and the Mechanisms of Fluorescence
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A Brief History of Fluorescent-Protein Research and the Mechanisms of Fluorescence
Author: 极地冰川
Reviewed by: 未名
Abstract
Green fluorescent protein (GFP), discovered and isolated from the jellyfish Aequorea victoria in 1962, and the red fluorescent protein drFP583, discovered in 1999 in corals of the genus Discosoma, are now among the most widely used proteins across biological research. They are nontoxic to organisms, efficiently emit characteristic fluorescence when excited by light of specific wavelengths, have low molecular weights, are readily incorporated into vectors, and can be expressed in many organisms. These properties give them broad potential in numerous fields. Many fluorescent proteins are already used in practical research.
History of Fluorescent-Protein Research
Green fluorescent protein was the first fluorescent protein to be discovered. In 1962, the Japanese scientist Osamu Shimomura first discovered and isolated GFP from the jellyfish Aequorea victoria. At first, it appeared as a by-product of Shimomura's extraction of aequorin. It looked green in sunlight, appeared intensely yellow under tungsten light, and fluoresced bright green under ultraviolet light. Through biochemical and spectroscopic studies, Shimomura made an initial determination of GFP's fluorescence mechanism and fluorophore[1]. Yet he did not realize that his discovery would transform modern biotechnology three decades later. Before long, he discontinued further work on GFP and moved into other areas of research.
Between 1985 and 1992, Douglas Prasher successfully determined the gene and protein sequences of GFP and, on that basis, cloned the GFP gene in 1992. Martin Chalfie of Columbia University was the first to use GFP in biological research. In 1994, Chalfie used molecular biology techniques to introduce GFP cDNA into model organisms, including nematodes, and successfully expressed it, revealing GFP's enormous potential as a reporter gene. That same year, Roger Tsien's team elucidated GFP's fluorescence mechanism and modified GFP through mutation, producing variants with greater fluorescence intensity, a single absorption peak, and more efficient conformational folding, such as GFP-S65T[2].
In 1999, a second type of fluorescent protein after GFP was first reported: the red fluorescent protein drFP583. Matz and his colleagues purified this tetrameric protein from corals of the genus Discosoma in the Pacific; it fluoresces red under ultraviolet illumination. Its long emission wavelength, high sensitivity, and high signal-to-noise ratio made it a valuable complement to GFP-based studies in vivo. The shortcomings of drFP583 itself, however—including oligomerization, slow maturation, and cellular toxicity—limited its use. In practice, researchers now more often use E57-NA, an engineered mutant produced by Clontech and sold under the name DsRed, which has lower cytotoxicity, a reduced tendency to oligomerize, and faster maturation. As DsRed has continued to be modified and improved, its range of applications has grown. In combination with GFP, it provides a new and rapid detection method for research in molecular biology, cell biology, and related fields[3].
How Fluorescent Proteins Produce Fluorescence
Fluorescent-Protein Structure
Structure of Green Fluorescent Protein
The primary structure of wild-type green fluorescent protein has been inferred from its corresponding cDNA sequence. Its primary sequence contains 238 amino-acid residues, and its molecular mass is approximately 27 kDa[2]. Analysis of X-ray diffraction images from prepared crystals[4] shows a cylindrical secondary structure approximately 4 nm high and 3 nm in diameter.
The wall of the cylinder consists of 11 β-strands wrapped into a barrel. The β-strands pack tightly against one another and form regular bands of hydrogen bonds. A short α-helix caps one end, producing a densely packed structure with a single opening known as a β-barrel.
At the same time, the α-helix that closes one end extends inward along the central axis and serves as a scaffold for the chromophore. The chromophore lies at the core of the structure, protected by the surrounding β-sheet layers. This architecture makes the overall structure highly stable and greatly increases the central chromophore's resistance to heat, acids, bases, denaturants, and other factors[5]. The same compactness, however, makes it difficult for enzymes or cofactors to enter GFP and catalyze chromophore formation. The chromophore must therefore form by autocatalysis.
The GFP chromophore forms from the Ser–Tyr–Gly triad at residues 65–67. Autocatalytic cyclization of the triad produces the 4-(p-hydroxybenzylidene)imidazolin-5-one structure that constitutes GFP's central chromophore.
Chromophore formation[7] is an autocatalytic cyclization process that requires no enzyme or cofactor. Ser65 and Gly67 first cyclize rapidly to form an imidazolin-5-one intermediate. O2 then slowly oxidizes the Tyr66 side chain to form a double bond. This slow step often takes several hours. Gly67 is essential for chromophore formation; no other amino acid can replace it. The reaction is also temperature-sensitive, and its yield falls above 30 ℃. This may be related to the low temperature of the jellyfish's natural habitat. Once formed, the chromophore itself is highly resistant to heat and is thermally stable.
Experiments have shown[7] that amino acids 2–232 in GFP's primary structure are required to maintain fluorescence. Truncating more than seven amino acids from the C terminus or more than two from the N terminus completely eliminates fluorescence. The final seven residues are disordered and do not form part of the protein's specific structure, so deleting or adding residues in this region does not affect its overall function. The N-terminal residues help form the “cap” at one end of the β-barrel. This indispensable structure protects the chromophore, though extending the peptide chain at the N terminus does not disrupt the protein. When GFP forms a fusion protein with certain other proteins, the loss of some C-terminal residues does not eliminate fluorescence, although it still affects the spectral properties to some extent[5].
GFP can dimerize in crystals or in solutions with ionic strength below 100 mmol／L. Dimerization facilitates energy transfer between GFP and the jellyfish photoprotein under natural conditions.
Structure of Red Fluorescent Protein (DsRed)
DsRed consists of 225 amino-acid residues and has a relative molecular mass of approximately 25.9 KDa. This coral fluorescent protein has little primary-sequence homology with GFP from Aequorea victoria—only about 23%. Their secondary structures, however, are highly similar. Like GFP, DsRed forms a β-barrel.
The DsRed chromophore is formed from Gln66–Tyr67–Gly68 and is structurally very similar to the GFP chromophore. During in vitro maturation, its initial excitation and emission wavelengths are 475 nm and 499 nm, respectively, producing green fluorescence. The green fluorescence reaches maximum intensity after about 7 hours and then begins to decay. After 2 days, the green fluorescence disappears completely, after which red fluorescence slowly begins to emerge. Analysis shows that formation of the DsRed chromophore first requires a structure essentially identical to the GFP chromophore, followed by further oxidation to form the DsRed chromophore. Compared with its GFP counterpart, the DsRed chromophore contains an imine group[9]. The imine double bond further extends the chromophore's conjugated system, shifting its emission toward longer wavelengths.
Crystallographic analysis shows that DsRed normally exists as a tetramer. Experiments further indicate that its chromophore can form and function only after oligomerization. Alkaline, acid, and guanidine denaturation experiments show that the DsRed chromophore forms through several folding steps and that oligomerization plays an important role in maturation[3].
Mechanism of Fluorescence
Existing research has established that the fluorescent proteins discovered so far produce fluorescence through broadly similar mechanisms. GFP was the first such protein discovered and studied, and research on its mechanism is now relatively mature. This section therefore focuses on how GFP produces fluorescence.
In Aequorea victoria, fluorescence from GFP is coupled to another luminescent protein, aequorin.
Aequorin uses coelenterazine as a prosthetic group and, in the presence of calcium ions, can emit blue light at 460–470 nm.
In the presence of calcium ions, aequorin is oxidized by oxygen and emits blue light. Through Förster resonance energy transfer, this energy reaches the GFP chromophore, which is excited and then emits GFP's characteristic green fluorescence at 509 nm.
GFP fluorescence is now generally believed to involve a process called excited-state proton transfer (ESPT)[11]. The key lies in the phenolic group on the Tyr66 side chain in the chromophore. Because a phenol is weakly acidic, it interconverts between neutral and ionic forms. Under ordinary conditions, the neutral form predominates, and the two occur in a ratio of approximately 6:1. However, when the chromophore
is excited by light, the acidity of the group increases greatly. Changes in the hydrogen-bond network formed jointly by amino-acid side-chain groups and by those side chains with water molecules in the chromophore's microenvironment play a decisive role in this process[12].
Crystallographic analysis shows that, in the unexcited state, the Tyr66 phenolic hydroxyl is neutral. Electrostatic repulsion from the Glu222 carboxyl group, together with hydrogen bonds involving the bound water molecule Wat304 and the side-chain oxygen of Ser205, maintains this state. The interaction between Glu222 and Ser65 stabilizes it further.
Upon excitation, Glu222 transmits a negative charge to the chromophore's phenolic hydroxyl through the hydrogen-bond network formed with Wat304 and Ser205, while the hydroxyl proton is transferred away. In most cases, the chromophore that accepts the charge undergoes photoisomerization and then returns to the ground state by emitting radiation at 504 nm, after which it becomes neutral again.
In a minority of cases, however, changes in the hydrogen-bond network stabilize the chromophore in its ionic form. A new network forms: the His148 side chain hydrogen-bonds to the Tyr66 phenolic oxygen (Oη), while Wat304 interacts with Tyr66 Oη and the side-chain oxygens of Thr203 and Ser205, thereby stabilizing the chromophore. At the same time, the hydrogen bond between Glu222 and Ser205 breaks, and the carboxyl oxygen of Glu222 may undergo cis–trans isomerization.
Engineering Fluorescent Proteins
Wild-type fluorescent proteins have several shortcomings, including low fluorescence intensity, temperature-sensitive folding, delayed fluorescence, and, for drFP583, cytotoxicity. Most fluorescent proteins used today have therefore been engineered. Such engineering generally centers on the chromophore and follows two basic approaches. The first directly alters the chromophore chemically, thereby changing its photochemical properties. The second replaces amino acids in the chromophore's microenvironment, using the altered surroundings to modify fluorescence. Several common engineered fluorescent proteins are introduced briefly below[11].
Enhanced Blue Fluorescent Protein (EBFP)
In wild-type GFP, Tyr66 is replaced with His, substituting an imidazole group for the original phenolic group. This mutation blue-shifts the excitation and emission spectra relative to GFP (excitation wavelength: 383 nm; emission wavelength: 445 nm). Val150 is also mutated to Ile and Val224 to Arg, filling the cavity left by replacing tyrosine with histidine and increasing chromophore stability.
Enhanced Cyan Fluorescent Protein (ECFP)
Cyan fluorescent protein follows the same engineering strategy as blue fluorescent protein. Replacing Tyr66 with tryptophan introduces an indole group into the chromophore. Its excitation and emission wavelengths then fall between those of the neutral and ionic phenolic forms of wild-type GFP, producing characteristic blue-green fluorescence (Ex: 439 nm; Em: 476 nm). In addition, replacing His148 with aspartic acid eliminates the interaction between Tyr145 and His148, stabilizing the protein's first conformational state. Replacing Tyr145 with Ala further increases the mutant's fluorescence intensity. The S72A mutation plays an important role in accelerating chromophore maturation.
Enhanced Yellow Fluorescent Protein (EYFP)
EYFP was produced mainly by engineering the microenvironment around the wild-type GFP chromophore. The largest change relative to avGFP is the replacement of Thr203 with tyrosine. When the fluorescent protein folds, the Tyr203 phenolic group extends into the internal cavity and forms a π–π stacking interaction with the chromophore's phenolic group. This mutation stabilizes the excited-state dipole moment of the chromophore, red-shifting both the absorption and emission spectra of GFP by approximately 20 nm. The Gln69-to-Lys mutation also increases chromophore ionization and shifts the emission wavelength another 1–2 nm toward the red.
Superfolder GFP
Superfolder GFP combines nine point mutations: S30R, Y39N, F99S, N105T, Y145F, M153T, V163A, I171V, and A260V. Compared with wild-type GFP, Superfolder GFP is more stable, has more efficient folding kinetics, can fold efficiently at 37 ℃, and is brighter than EGFP. It also tolerates acids and bases better than other members of the GFP family.
Orange Fluorescent Protein (mOrange)
mOrange belongs to the mFruit family of fluorescent proteins and has characteristic absorption and emission wavelengths of 548 nm and 562 nm, respectively. Engineered from the red fluorescent protein DsRed, the mFruit family has the relative advantages of being monomeric, having low toxicity and high brightness, and maturing quickly. mOrange fluoresces orange because an oxazole ring forms at Thr66 during chromophore formation. Formation of the oxazole ring reduces the conjugation of the carbonyl group at residue 65, blue-shifting the absorption and emission spectra relative to the wild type.
Commentary
From the single green fluorescent protein first discovered to today's diverse range of proteins with different spectral properties, fluorescent proteins can fairly be said to have transformed biological research. They have given life scientists exceptionally effective tools and marked a turning point for the field. As excellent new labels, fluorescent proteins are now widely used throughout biology. Further research will undoubtedly open still broader prospects for their application.
Main References
[1] Zhu Yun, Li Chaowei. Green Fluorescent Protein[J]. Biology Teaching, 2010, 35(6): 62.
[2] Wang Yupeng. The Story of Fluorescent Proteins[J]. Biology Teaching, 2019, 44(5): 73-74.
[3] Hao Limei, Li Tangdi. Advances in Red Fluorescent Protein Research[J]. Foreign Medical Sciences: Pharmacy, 2006, 33(2): 131-133.
[4] Liu Mofang, Wang Enduo. Green Fluorescent Protein[J]. Progress in Biochemistry and Biophysics, 2000, 27(3): 238-243.
[5] Liu Zuqiang, Hu Min, Qi Yipeng. The Structure, Fluorescence Mechanism, and Applications of Green Fluorescent Protein[J]. Journal of Wuhan University, 2000, 46(2): 211-214
[6] Remington, S.J., Saif, M. Crystal structure of GFP S205A/T203V at 2.2 A resolution [DB/OL]. www.pdbus.org/structure/4OGS, 2014-05-14/2021-12-20.
[7] Ma Jinshi. Green Fluorescent Protein[J]. Chemistry Bulletin, 2009, 3: 243-250.
[8] Tubbs, J.L., Tainer, J.A., Getzoff, E.D. High Resolution Crystal Structure of the Discosoma Red Fluorescent Protein (DsRed) [DB/OL]. www.pdbus.org/structure/1ZGO, 2005-08-02/2021-12-20.
[9] Fan Jinyu, Cui Zongqiang, Zhang Xian’en. Spectral Diversity and In Vitro Molecular Evolution of Red Fluorescent Proteins[J]. Progress in Biochemistry and Biophysics, 2008, 35(10): 1112-1120.
[10] Head, J.F., Inouye, S., Teranishi, K., Shimomura, O… Crystal Structure of Aequorin[DB/OL]. www.pdbus.org/structure/1EJ3, 2000-05-31/2021-12-21.
[11] Sample V, Newman R H, Zhang J, et al. The structure and function of fluorescent proteins[J]. Chemical Society Reviews, 2009, 38(10): 2852-2864.
[12] Wang Wei, Liu Xianglin, Li Jiudi. The Fluorescence Mechanism of Green Fluorescent Protein[J]. Chemistry of Life, 1999, 19(6): 275-279.
Author: 极地冰川
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