Green

Fluorescent Protein: A Study

Matt Brown and Cameron Kubota

Fluorescent Proteins are members of a structurally homologous class of proteins

that share the same unique properties. These proteins contain a characteristic chain of three key amino acid residues that together, compose the vital chromophore that allows for the fluorescent proteins fluorescent nature upon excitation by a photon. Green Fluorescent Protein is just one of the many types of fluorescent proteins. History The first written record of bioluminescence was by Pliny the Elder in the first century AD, who observed glowing jellyfish in the Bay of Naples.1 In 1962, Osamu Shimomura, a Japanese organic chemist and marine biologist who was working with Princeton University, found out more about how this fluorescence was made. When studying the aequorin protein of Aequorea Victoria jellyfish what were collected at the Friday Harbor Laboratories of the University of Washington, Shimomura described GFP for the first time. Although he was mainly focused on aequorin he described, a protein giving solutions that look slightly greenish in sunlight through only yellowish under tungsten lights, and exhibiting a very bright, greenish fluorescence in the ultraviolet of a Mineralite, has also been isolated from squeezates.2 He went on to study GFP more and describe the protein in more detail. In 1979 Shimomura proposed the first chemical structural model of the chromophore of Aequorea GFP. The mechanics of renaturation of GFP, its spectral properties, external factors affecting its fluorescence, and many other properties of were studied subsequently, although it remained an obscure protein. Research on the protein was revolutionized after Martin Chalfies discoveries in 1994. He showed that that GFP could be used to monitor gene expression and protein localization in both prokaryotic and eukaryotic cells.3 Because GFP need no exogenous substrates or cofactors to fluoresce it became

a highly studied and used protein. In 1996, two groups published about the crystal structure of GFP.4,5 After Chalfies contribution, the research has shifted toward the applications of GFP and the preparation of mutants. Robert Tsien furthered research with articles about how to improve GFP fluorescence and photostability. In 2008 The Nobel Prize in Chemistry was awarded to Shimomura, Chalfie, and Tsien for their pioneering work in the discovery and development of green fluorescent protein.6

Importance of GFP Green Fluorescent Protein has revolutionized various fields of cell biology and

remains an integral part in the advancing of biological imaging, taking full advantage of its fluorescent properties. Scientists can splice GFP into the genome of specific, targeted cells. Because GFP is now part of the genomic sequence of the tagged cell, when the cell divides and reproduces, GFP is also replicated, resulting in the presence of GFP wherever the tagged cell is also present. This property of GFP and other fluorescent proteins is utilized in the phenomenon scientists refer to as Brainbow. Because these fluorescent proteins continuously reproduce along with the targeted cell, in 2007, Jean Livet of Harvard University spliced into various genomes the codes for different fluorescent proteins. The
Figure 1. Brainbow (from Livet, J. The Brain in Color: Transgenic Brainbow mice for visualizing neuronal circuits, 2007)

targeted cells were all cerebral cells from a mouse brain and upon photon excitation, the fluorescent proteins that were infused in the coding of the cerebral cells emitted their

respective colors, resulting in a brilliantly mapped brain.7 This useful trick allows for the 2

analysis of brain circuitry and the structural mapping of the brain. Although the fluorescent properties of the Green Fluorescent Protein allows for these extraordinary uses, the actual use of wild type GFP (wtGFP) in the Aequorea Victoria is unknown. Why is it important the color the jellyfish emits is green? Why does it have to convert the blue emission from the Aequorin photoprotein to the green emission through GFP? These are just a few of the questions scientists are looking to answer. Structure Green fluorescent protein has 238 residues and a beta-barrel structure, with

disordered turns and helical sections closing off the ends. GFP is almost a perfect cylinder with a height of 42 Angstrom and a diameter of 24 Angstrom.5 A central helix contains the chromophore, three cyclized residues that cause the protein to fluoresce. There is a matrix of hydrogen-bonds inside the beta barrel that keep the cyclized chromophore in the right alignment for fluorescence.6 The folding of GFP is essential for it to maintain fluorescence. GFP exhibited a new

protein fold when observed by Yang et al in 1996, a beta-barrel encircling a single central helix, and they dubbed the new fold a beta-can. There are 11 beta-strands in the barrel, and each strand is 9-13 residues long.5 All of the strands are parallel except for one interaction between the first and sixth strands. Starting at the N-terminal end, there is a short helical section, followed by three antiparallel beta strands. Next comes the central helix going up the center of the barrel containing the chromophore, followed by two smaller helical sections on the top. Then the polypeptide makes three more beta strands, the last of these three has a parallel interaction with the very first beta strand of the protein. After this, the 3

protein takes a turn on the bottom and makes a Greek key motif with the last five beta strands.5 The helical sections and the loops on the top and bottom of the barrel seal off and protect the chromophore inside from ligands or oxygen that could quench the fluorescence of the protein.4 The main chain hydrogen bonding lacing the surface of the cylinder likely accounts for the unusual stability of the protein toward denaturation and proteolysis.5 The folding structure of GFP is essential in order to protect the chromophore and maintain its fluorescent function, despite whatever environmental conditions it may encounter. The surrounding beta-barrel also interacts with the chromophore. Some residues in

the structure provide physical support while others help with the chemistry needed for fluorescence. The chromophore is relatively perpendicular to the long axis of the beta- barrel, it is at 60.5 The chromophore is a two-ring structure and needs to be held with the rings in the same plane in order to fluoresce. If the two rings are allowed to be perpendicular to each other, they give of thermal energy rather than radiate light energy.7 This means that the
Figure 2. A. Shows the overall structure of GFP with a beta-can protecting the chromophore inside. B. Shows how the residues fold. (Ormo, Crystal Structure of the Aequorea victoria Green Fluorescent Protein, 1996).

chromophore needs an enclosing cavity that is

very close fitting. Experiments have demonstrated reduced fluoresce in mutant fluorescent proteins that have different cavity shapes.7 On one end of the chromophore, the cavity is 4

slightly larger, maybe because it needed more space during chromophore formation. Fortunately, this extra space is filled with four water molecules that hydrogen bond with the chromophore as well as Glu222 and Gly69. Without these hydrogen bonds, the protein would be expected destabilize slightly.5 The other side of the chromophore many polar interactions take place. His148, Thr203, and Ser205form hydrogen bonds with the phenolic hydroxyl; Arg96 and Gln94 interact with the carbonyl of the imidazolidinone ring, and Glu222 forms a hydrogen bond with the side chain of Thr65.5 Arg96 and Glu222 have been shown to be catalytic, which means that they are very important, but not essential for chromophore formation. When GFP is fluorescing these residues that come in close contact with the chromophore are essential in changing the chromophore back and forth from anionic to neutral.7 The formation of the chromophore is a unique part of the structure of GFP because

the chromophore can be made without the aid of outside cofactors or enzymes. This allows GFP to be fluorescent by just splicing the genetic code of GFP into the genome of other organism, without needing any additional tampering with the organisms DNA or the addition of foreign cofactors. In wild-type GFP the chromophore is made of Ser65, Try66, and Gly67 which autocatalyze into a ring. The chromophore matures by cyclizing post- translation to become 4-(p-hydroxybenzylidene)- imidazolidin-5-one.4 There is evidence for a sequential model of chromophore formation.2 The three steps in the process of autocatalyzation are cyclization, dehydration, and oxidation. First, GFP folds into an almost native position by the Ser65 and Try66 flipping and the Ser66 rotating to be more accessible to Gly67.2 This three-dimensional shift is a driving force in the cyclization by placing the amino group of the Gly67 near the carbonyl group of 5

Try66.1 Then the amide group of Gly67 does a nucleophilic attack on the carbonyl carbon of Try66 to cyclize and form the imidazolidin ring. This is similar to Asn-Gly cyclization that has been observed before.2 It is essential to have Gly as residue 67, because its lack of steric hindrance makes cyclization possible. Gly is conserved in all forms of GFP and is essential for proper formation of the
Figure 3. Shows the steps in the autocatalyzation of the chromophore. Top row, left to right, shows the protein movement. Bottom row, left to right, shows cyclization, dehydration, and oxidation to produce the mature chromophore. (Day, Richard et. al., Aequorea Victoria Fluorescent Proteins, http://zeiss- campus.magnet.fsu.edu/articles/probes/jellyfishfps.html)

chromophore.2 The next step of chromophore formation is dehydration, where water comes off of Ser65, leaving behind an imidazolin-5-one intermediate.1 The last step is reduction of the alpha-beta bond of Try66 by molecular oxygen.2 GFP grown anaerobically in Escherichia coli do not fluoresce because they do not have molecular oxygen needed to perform this last step.1 When oxygen is re-administered the GFP gains fluorescence. By examining the rate at which GFP gains fluorescence as well as studying electrospray ionization mass spectroscopy of GFP the conclusion is that GFP can cyclize and dehydrate without oxygen present.1 This is strange that molecular oxygen is needed in the formation of the chromophore, because after it is formed, oxygen will quench its fluorescence. Even once it is made, the chromophore needs to be surrounded by the rest of the protein in order to fluoresce. Both water and oxygen will quench its 6

fluorescence, so it needs the protection of the beta barrel. The naked chromophore is neither rigid nor protected from jostling by solvent molecules so it cannot yet fluoresce.1 Another physical characteristic of GFP is its fluorescence. GFP has a major peak of absorption at 375nm in the UV range, and a minor peak at 475nm which is the green visible light that we see. When excited at 395nm, emission peaks at 508nm.2 These two peaks in absorbance at 375nm and 479nm are due to the two different states the chromophore can

take, either neutral (A form) or anionic (B form). The neutral chromophore absorbs at 395nm, while the anionic form absorbs at

Figure 4. The absorbance peaks at 395nm and 475nm and the chromophore structures that cause them. A is the neutral and B is the anionic form of the chromophore. (Remington, Green Fluorescent Protein: A perspective, 2011.)

475nm. In wild-type GFP, these are always seen in a 6:1 ratio, favoring the neutral. This is because the structure of GFP effectively protects the chromophore from outside influences, keeping the interactions with the chromophore very predictable to preserve the ratio. By mutating key residues, this ratio can be changed or the absorption peaks can be changed to fluoresce different colors or at different intensities. The structure of GFP leads to its characteristic fluorescence. The structure of green fluorescent protein is essential for proper protein function. For example, the beta-barrel protects the chromophore, keeping it in the proper conformation to fluoresce. The chromophore is also assisted by other residues to go from neutral to anionic. Without its specific structure this protein would not be fluorescent or would not nearly as useful as it is as a biological marker. 7

Function/Direction of Research The main biological function of GFP is to emit a green photon at 509 nm upon the

excitation of the chromophore. This overall function of GFP occurs only after a series of events take place, a series of events that depend on the type of GFP being studied. For example, while in wtGFP, a calcium ion must activate the Aequorin photoprotein, which in turn will release a photon of blue light with an emission peak at 470 nm that can activate GFP, in vitro studies utilize UV light for the
Figure 5. The biological mechanism for the ultimate emission of the green photon in wtGFP (from The GFP Site, http://gfp.conncoll.edu/)

emission of the photon with the capability of activating GFP. Since GFP has two excitation peaks, one major peak at 396 nm and a minor peak at 475 nm, research suggests that there is more than one way to excite the photoprotein and result in the emission of the green light. This green light emission is principally and directly the product of the Green

Fluorescent Protein chromophore. The chromophore is naturally found in the p-HBDI conformation, but can also be forced into its o-HBDI conformation at no loss of fluorescence.8 The chromophore is composed of Serine 65, Tyrosine 66, and Glycine 67 which autocatalyze to form the mature GFP chromophore, as discussed earlier. This GFP Chromophore is a strong photoacid as upon the excitation of the chromophore by the photon, it takes on acidic properties. This occurrence was observed when upon photon absorption the pKa of the chromophore changed from 8.0 to 1.0.9 This acidic nature of the chromophore will later play a key role in the deprotonation of the chromophore, an event 8

that ultimately leads to the fluorescence emitted from the mature chromophore. However, in order to become deprotonated, the chromophore must naturally hydrogen bond with other key residues. In fact, the GFP chromophore is interconnected to many other residues both on the -helix and a part of the -barrel. The network of hydrogen bonding both stabilizes the chromophore, retaining the

integrity of the chromophore by ensuring it remains planar, and most importantly, it facilitates the chain of proton transfers from the chromophore to surrounding residues. The chain of hydrogen bonds that specifically participate in the excited state proton transfer is the relay from the chromophores Tyrosine 66 residue to a water molecule (often numbered residue 285), to Serine 205, and finally to Glutamic Acid 222.6 Excited State Proton Transfer, or ESPT for short, is
Figure 6. The network of hydrogen bonding in which the chromophore participates (from The Institute of Bioorganic Chemistry of the Russian Academy of Sciences, http://www.ibch.ru/en/structure/groups/xray)

activated by the absorption of a photon. This photon is absorbed solely at the 396 nm wavelength and triggers a cascade of events which ultimately results in the deprotonation of the Tyrosine 66 residue and the protonation of the Glutamic Acid 222 residue. Upon the extraction of GFP from the Aequorea victoria, scientists found that the neighboring photoprotein, called Aequorin, was responsible for releasing this blue colored photon when activated to do so by a Calcium ion. This blue photon excites the GFP chromophore allowing for wild-type GFP to emit its green fluorescence. Figure 7 shows the absorbance of the photon of light (denoted hv) by the A state of the chromophore. As depicted and described 9

earlier, the photoacidic properties of the chromophore result in an acidic chromophore once it absorbs the blue emission. Due to its acidic properties, absorbance of the photon and the excitation of the chromophore causes Tyrosine 66 to become deprotonated, transferring its proton to the neighboring hydrogen-bonding partner, the water molecule. This is the rate-determining step, and once the water molecule is protonated, the subsequent deprotonation of the water molecule to the protonation and deprotonation of the Serine 205 residue and finally the protonation of the Glutamic Acid
Figure 7. Excited State Proton Transfer Mechanism (from Remington, S. James, Green Fluorescent Protein: A Perspective, 2011)

222 residue all occur within picoseconds.10 The deprotonation of the chromophore and the

ultimate protonation of the Glutamic Acid 222 residue produce the I*-state of the chromophore, or the excited intermediate state. This is the state of the chromophore that releases the green fluorescence. Once the I*-state begins to lose its energy to the emission of the green photon, the I* state relaxes and converts back into the I state, or the stable intermediate state, and then returns to the A-state of the chromophore by the deprotonation of the Glutamic Acid 222 residue and the residual re-protonation of Tyrosine 66.6 The other equilibrated state that the chromophore can take on is called the B-state,

or the anionic state. This state is not formed very often as was shown in the 6:1 ratio between the A and the B states of the chromophore. However, this state does exist and is a 10

simple conformation change of the Glutamic Acid 222 residue from the I state, the I state being the anti conformer and the B state being the syn conformer. In the B state, because the syn conformation prevents the hydrogen bond from being formed between the Glutamic Acid 222 residue and the Serine 205 residue, there is no excited state proton transfer that will result in the fluorescence of the green photon. Instead, this state may also be excited when it absorbs a photon at 475 nm and changes into the excited B state, B*- state, which also emits the green fluorescing photon. Whereas I*-state is indirectly excited when the A-state becomes excited, resulting in the emission of the green fluorescence, the B state excites and fluoresces directly, resulting in a more efficient fluorescence.11 Just

recently, interest in GFP has spiked, a prime example of this increase was the awarding of the 2008 Nobel Prize in Chemistry to Roger Tsien, Martin Chalfie, and Osamu Shimomura for the discovery and development of GFP. However, the natural state of GFP was not good enough for many scientists. Over the past decade, much of the research done regarding Green Fluorescent Protein has been observing the results of mutations on the structure and function of GFP. One important thing to remember about any protein however is that structure determines function and because of this, mutations must result in a conservation in packing and folding, otherwise the mutation will be detrimental to the protein and the green fluorescence properties of GFP will disappear. One of the most important and biggest discoveries in the field of mutations to GFP was the substitution of Threonine for Serine 65. In 1995, upon the observation that the 475 nm excitation fluorescence had a good photostability, even greater than the fluorescence that resulted from the 396 excitation peak, but was relatively low in amplitude, Roger Tsien, Roger Heim, and Andrew Cubitt set out to improve the fluorescence resulting from a 475 nm excitation. They tested many 11

different amino acids such as Alanine, Leucine, Cysteine, and Threonine to see if, like Serine, excitation of the mutated chromophore would produce a vinyl side chain and either H2O or H2S (in the case of Cysteine). To their surprise, each of these amino acid mutations procured the desired results, but they decided that the most practical mutation was the substitution of Threonine for Serine 65 (S65T), because the Threonine mutant resulted in the longest wavelength of excitation and the highest relative amplitude upon excitation by an external photon. In Figure 9, the relative amplitude of
Figure 8. Fluorescence observed in GFP extracted from cDNA (left). Fluorescence observed in S65C mutation (right) (from Reichel et. al., Transient Expression of a Mutated GFP cDNA Leads to Improved Fluorescence in Plant Protoplasts, 1996)

the mutated S65T is significantly higher than the emission peak of the wild type GFP.12 Although the S65T mutant results in stronger fluorescence, one oddity that the figure presents shows up in the representation of the excitation peaks. In wild type GFP, there are two clear excitation peaks, one at around 396 nm and the
Figure 9. Excitation (dashed lines) and emission (solid lines) spectra of wt GFP (gray lines) and GFP-S65T (black lines), (from Kain, Steven R. et. al., Expression and Detection of Green Fluorescent Protein (GFP), 1997)

other at around 475 nm. This would be expected due to both forms of the chromophore can take on, either A or B state, that in its own way can result in

the fluorescence of the protein. However, in the S65T mutant, there is only one excitation peak around the 475 nm mark instead of the normal two peaks. In 1997, further investigation found that the alteration of Serine 65 to Threonine restricts the excited state

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proton transfer from the chromophore to Glutamic Acid 222 and instead, fluoresces solely through the excitation of the B-state.11 Another mutation that is similar in nature to the S65T mutation is the replacement

of Glutamic Acid 222, the final protonated residue in the series of excited state proton transfers, with Glutamine. Because Glutamine doesnt possess the same acidic properties that Glutamic Acid has, Glutamine cant participate in excited state proton transfer, thus also eliminating fluorescence through the excitation of the A-state, solely producing green emission through the excitation of the B-state. This mutant is referred to as the E222Q mutant.13 With the discovery that the S65T and E222Q mutants restricted fluorescence of the

green fluorescent protein through the A-state and excited state proton transfer, a group of scientists in 2008 became determined to find a way to recover this loss of fluorescence. Their answer? Replace Histidine 148 with Aspartic Acid. This mutation in both the case of the S65T mutant and the E222Q mutant recovered its absorbance at the 396 nm wavelength, representing a recovery of excited state proton transfer. However, the proton wasnt transferred
Figure 10. A) Overall series of hydrogen bonds connecting the chromophore to E222 residue. B) Hydrogen bonds in which Tyrosine 66 and water participate (from Shinobu, Al et. al., Visualizing Proton Antenna in a High-Resolution Fluorescent Structure, 2011)

normally, through water and Serine 205 to Glutamic Acid 222, but rather, straight to the Aspartic Acid 148 residue. This discovery proved that there is more than one pathway for

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the proton to travel which allows for the deprotonation of the chromophore and the recovery of the green fluorescence through the I*-state and indirectly, the A-state.11 Another important mutation that was discovered was the substitution of Histidine

for Threonine 203, resulting in what is called the photoactivatable GFP or PA-GFP. While the S65T and E222Q mutants halt the excitation of the chromophores A-state at 396 nm, the T203H mutant promotes the excitation of the A-state chromophore and instead inhibits fluorescence through the B-state of the chromophore. This means that the photon excitation peak at 475 nm for wild type GFP doesnt exist
Figure 11. Absorption and Fluorescence emission spectra of native PA-GFP, photoactivated GFP, and wild-type GFP (from Henderson, J. Nathan et. al., Structure and Mechanism of the Photoactivatable Green Fluorescent Protein, 2008)

for this mutant and the peak at 396 nm is amplified, just like how

for the S65T mutant, the 475 nm excitation peak was amplified and the 396 nm excitation peak was gone altogether (See Figure 11. Note the missing peak for PA-GFP at the 475 nm mark). It was determined that the reason for the loss of absorption through the B-state is that upon photoactivation of the wild-type GFP, Threonine 203 actually rotates and repositions itself to hydrogen bond with the anionic-B-state, further stabilizing the formation of the B-state chromophore. By swapping out Histidine for Threonine 203, this rotation is not an option and Histidine 203 remains unaltered upon photoactivation in PA- GFP.14

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This trend of structurally altering GFP and its chromophore and studying the effects

of the alteration on the function of GFP has continued even to today. Published in the Journal of the American Chemical Society on February 16, 2011, scientists forced the naturally forming p-HBDI (the GFP chromophore) to
Figure 12. Structure of p-HBDI and o-HBDI (from Hsieh, Cheng-Chih et. al., Comprehensive Studies on an Overall Proton Transfer Cycle of the ortho-Green Fluorescent Protein Chromophore, 2011)

mature as o-HBDI, a slightly different conformation. Structurally, ortho-HBDI is very

similar. The only difference is the intrinsic seven-membered ring, one of the bonds being made by a hydrogen bond between the O-R group and the nitrogen of the imidazoline ring, as seen in Figure 12. This means that the Tyrosine 66 residue is not hydrogen bonded to either the water molecule or H148 and can not pass its proton off through excited state proton transfer normally. However, when the o-HBDI conformer was excited in much the same way as the p-HBDI, naturally occurring GFP chromophore, is
Figure 13. Mechanism of o-HBDI (from Hsieh, Cheng-Chih et. al., Comprehensive Studies on an Overall Proton Transfer Cycle of the ortho-Green Fluorescent Protein Chromophore, 2011)

excited, it also resulted in fluorescence. After further studying of the exact mechanism of the o-HBDI conformer of the chromophore, shown in Figure 13, the study showed that instead of having to rely on outside residues for proton transfer in either the -barrel or the helix and chromophore maturation, it can instead undergo excited state intramolecular proton transfer. This means that the proton is transferred to bond with the nitrogen in the imidazoline ring and rearranges to form a tautomer intermediate. It is this 15

tautomer intermediate that allows for the fluorescence to occur when the chromophore is in the o-HBDI conformer, much like the how in the p-HBDI conformer, it is the intermediate state, I*-state, that fluoresces.15 For many years, scientists have been mutating and altering the structure of GFP and

its chromophore in order to determine key residues and how to improve the fluorescence of the chromophore. However, in 2010, a team of scientists experimentally observed the effects an ionic liquid would have on the structure and thermal stability of green fluorescent protein. In this study, GFP was placed in two different assays of the ionic liquid 1-butyl-3-methylimidazoline chloride, one with 25% and the other with 50% of the ionic liquid by volume. They observed that the ionic liquid interfered with the crystal structure of the green fluorescent protein structure as the dissolving of GFP in 50% by volume of the ionic liquid caused the protein to exist as a monomer rather than its naturally occurring dimeric state when dissolved in water. Another effect of the ionic liquid was that the protein appeared less compact in the ionic liquid than when it was dissolved in water, which implies that there was slight unfolding in the structure of the protein. This phenomenon became more apparent when the thermal stability of the protein in the ionic solution was tested. Figure 14 depicts the denaturation curves for GFP in various solutions, both the bottom curves belonging to the GFP dissolved in the ionic 16
Figure 14. Denaturation Curves for GFP measured in 10 mM TrisHCl (pH 7.4) (black), water (red), 25 vol % [bmim]Cl (blue), and 50 vol % [bmim]Cl (green) (from Heller, William et.al., Characterization of the Influence of the Ionic Liquid 1- Butyl-3-methylimidazoline Chloride on the Structure and Thermal Stability of Green Fluorescent Protein, 2010)

solutions, the blue line being the 25% by volume of the ionic liquid and the green line being the 50% by volume of the ionic liquid. In both cases, the GFP denatured and lost its fluorescent nature at a much lower temperature than we would have expected should the GFP had been dissolved normally in neat water. From this information, it was concluded that the ionic liquid the team had chosen to test, if not all ionic liquids, interfere with the crystal structure of the GFP protein and hinder its function greatly.16 Green Fluorescent Protein, being a relatively new molecule, has just recently

garnered attention for its phenomenal properties and the role it plays in cell imaging. Research in GFP, as evidenced by the numerous studies outlined, has been heading towards the observation and understanding of the effects of stressors on the structure and function of GFP, whether it be mutations or outside, environmental effects. Researchers have also been studying the ability for GFP to recover its fluorescence once restricted, like in the study of mutating Histidine 148 to Aspartic Acid. Also just recently, it was discovered that the GFP chromophore can actually be structurally altered to produce many other fluorescing proteins of differing colors, the most
Figure 15. GFP and its derivatives (from Remington, S. James, Green Fluorescent Protein: A Perspective, 2011)

common derivatives of GFP being the Red, Orange, and Yellow fluorescing proteins.6 Because of this, research has

also been starting to trail away from Green Fluorescent Protein specifically and more towards understanding the nature and properties of fluorescent proteins in general. However, Green Fluorescent Protein remains an integral aspect of science today, and through uses such as Brainbow, GFP continues to change the face of biological imaging. 17

Works Cited 1. Cubit, A.B et. al., Understanding, improving and using green fluorescent proteins. Trends in Biochemical Sciences. 1995;20 (11): 448-455 2. Tsien, R.Y.; The Green Fluorescent Protein. Annu. Rev. Biochem 1998;67: 509544 3. Chalfie, M.; Tu, Y.;Euskirchen, G.;Ward, W.W.;Prasher, D.C. Green fluorescent protein as a marker for gene expression. Science; Feb 11, 1994; 263, 5148. 4. Yang, F.;Moss, L.G.; Phillips, G.N.Jr. The molecular structure of Green Fluorescent Protein. Nature Biotechnology,1996, 14 (10), pp. 1246-1251. 5. Ormo, Mats; Cubitt, Andrew B; Kallio, Karen; Gross, Larry A; Tsien, R.Y.;Remington, S.J. Crystal structure of the Aequorea victoria green fluorescent protein. Science; Sep 6, 1996; 273, 5280. 6. Remington, S. J., Green Fluorescent Protein: A Perspective. Protein Science 2011;20: 15091519 7. Livet J. The brain in color: transgenic Brainbow mice for visualizing neuronal circuits. Med Sci (Paris) 2007;23:11731176. [in:French] 8. Kew-Yu Chen et. al. Ortho Green Fluorescence Protein Synthetic Chromophore; Excited-State Intramolecular Proton Transfer via a Seven-Membered-Ring Hydrogen-Bonding System. Journal of American Chemical Society 2007;129:4534- 4535 9. Tolbert, Laren et. al. Excited-State Proton Transfer: From Constrained Systems to Super Photoacids to Superfast Proton Transfer. Accounts of Chemical Research 2002; 35:19-27 10. Lill, Markus A. et. al., Proton Shuttle in Green Fluorescent Protein Studied by Dynamic Simulations. PNAS 2002;5: 2778-2781 11. Stoner-Ma, Deborah et. al., Observation of Excited-State Proton Transfer in Green Fluorescent Protein using Ultrafast Vibrational Spectroscopy. Journal of American Chemical Society 2004;9: 2864-2865 12. Tsien, Roger et. al., Improved Green Fluorescence. Nature 1995;373: 663-664 13. Jung, Gregor et. al., The Photophysics of Green Fluorescent Protein: Influence of the Key Amino Acids at Positions 65, 203, and 222. Biophysical Journal 2005;3: 1932- 1947 18

14. Henderson, J. Nathan et. al., Structure and Mechanism of the Photoactivatable Green Fluorescent Protein. Journal of American Chemical Society 2009;131: 4176-4177 15. Hsieh, Cheng-Chih et. al., Comprehensive Studies on an Overall Proton Trasnfer Cycle of the ortho-Green Fluorescent Protein Chromophore. Journal of American Chemical Society 2011;133: 2932-2943 16. Heller, William et.al., Characterization of the Influence of the Ionic Liquid 1-Butyl-3- methylimidazolium Chloride on the Structure and Thermal Stability of Green Fluorescent Protein. Journal of American Chemical Society 2010;114: 13866-13871

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