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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 protein’s 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 Chalfie’s 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
targeted cells.7 This useful trick allows for the 2 . the research has shifted toward the applications of GFP and the preparation of mutants. This property of GFP and other fluorescent proteins is utilized in the phenomenon scientists refer to as “Brainbow”. resulting in a brilliantly mapped brain. two groups published about the crystal structure of GFP.5 After Chalfie’s contribution. The Figure 1. and Tsien for their pioneering work in the discovery and development of green fluorescent protein. Robert Tsien furthered research with articles about how to improve GFP fluorescence and photostability. taking full advantage of its fluorescent properties. Chalfie.4. J. Jean Livet of Harvard University spliced into various genomes the codes for different fluorescent proteins. Because GFP is now part of the genomic sequence of the “tagged” cell. The Brain in Color: Transgenic “Brainbow” mice for visualizing neuronal circuits. Brainbow (from Livet. 2007) targeted cells were all cerebral cells from a mouse brain and upon photon excitation.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. resulting in the presence of GFP wherever the tagged cell is also present. in 2007. Scientists can splice GFP into the genome of specific. GFP is also replicated. In 1996. Because these fluorescent proteins continuously reproduce along with the targeted cell. In 2008 The Nobel Prize in Chemistry was awarded to Shimomura. when the cell divides and reproduces.a highly studied and used protein. the fluorescent proteins that were infused in the coding of the cerebral cells emitted their respective colors.
5 A central helix contains the chromophore. followed by three antiparallel beta strands. the last of these three has a parallel interaction with the very first beta strand of the protein. GFP is almost a perfect cylinder with a height of 42 Angstrom and a diameter of 24 Angstrom. Then the polypeptide makes three more beta strands. Next comes the central helix going up the center of the barrel containing the chromophore. There are 11 beta-‐strands in the barrel. 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. three cyclized residues that cause the protein to fluoresce. and each strand is 9-‐13 residues long. the 3 . the actual use of wild type GFP (wtGFP) in the Aequorea Victoria is unknown. There is a matrix of hydrogen-‐bonds inside the beta barrel that keep the cyclized chromophore in the right alignment for fluorescence. Structure Green fluorescent protein has 238 residues and a beta-‐barrel structure. a beta-‐barrel encircling a single central helix.analysis of brain circuitry and the structural mapping of the brain.6 The folding of GFP is essential for it to maintain fluorescence.5 All of the strands are parallel except for one interaction between the first and sixth strands. Starting at the N-‐terminal end. with disordered turns and helical sections closing off the ends. After this. Although the fluorescent properties of the Green Fluorescent Protein allows for these extraordinary uses. GFP exhibited a new protein fold when observed by Yang et al in 1996. and they dubbed the new fold a beta-‐can. followed by two smaller helical sections on the top. there is a short helical section.
1996). despite whatever environmental conditions it may encounter.7 This means that the Figure 2. chromophore needs an enclosing cavity that is very close fitting. If the two rings are allowed to be perpendicular to each other. Experiments have demonstrated reduced fluoresce in mutant fluorescent proteins that have different cavity shapes. The surrounding beta-‐barrel also interacts with the chromophore.7 On one end of the chromophore. Crystal Structure of the Aequorea victoria Green Fluorescent Protein. they give of thermal energy rather than radiate light energy.protein takes a turn on the “bottom” and makes a Greek key motif with the last five beta strands. Shows the overall structure of GFP with a beta-‐can protecting the chromophore inside.5 The folding structure of GFP is essential in order to protect the chromophore and maintain its fluorescent function. (Ormo. The chromophore is relatively perpendicular to the long axis of the beta-‐ barrel. B. it is at 60°.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”. Some residues in the structure provide physical support while others help with the chemistry needed for fluorescence. Shows how the residues fold. A. the cavity is 4 .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.5 The chromophore is a two-‐ring structure and needs to be held with the rings in the same plane in order to fluoresce.
which means that they are very important. Without these hydrogen bonds.4 There is evidence for a sequential model of chromophore formation. maybe because it needed more space during chromophore formation.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 . Try66.7 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.2 The three steps in the process of autocatalyzation are cyclization. without needing any additional tampering with the organism’s DNA or the addition of foreign cofactors. but not essential for chromophore formation. and oxidation. Fortunately. and Glu222 forms a hydrogen bond with the side chain of Thr65”. and Ser205form hydrogen bonds with the phenolic hydroxyl. this extra space is filled with four water molecules that hydrogen bond with the chromophore as well as Glu222 and Gly69. 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. First. In wild-‐type GFP the chromophore is made of Ser65. Thr203. Arg96 and Gln94 interact with the carbonyl of the imidazolidinone ring. This allows GFP to be fluorescent by just splicing the genetic code of GFP into the genome of other organism. and Gly67 which autocatalyze into a ring. “His148.slightly larger. GFP folds into an almost native position by the Ser65 and Try66 flipping and the Ser66 rotating to be more accessible to Gly67. the protein would be expected destabilize slightly. dehydration. The chromophore matures by cyclizing post-‐ translation to become 4-‐(p-‐hydroxybenzylidene)-‐ imidazolidin-‐5-‐one.5 Arg96 and Glu222 have been shown to be catalytic.5 The other side of the chromophore many polar interactions take place.
shows the protein movement. Gly is conserved in all forms of GFP and is essential for proper formation of the Figure 3. dehydration. Both water and oxygen will quench its 6 .2 GFP grown anaerobically in Escherichia coli do not fluoresce because they do not have molecular oxygen needed to perform this last step. because its lack of steric hindrance makes cyclization possible. al. This is similar to Asn-‐Gly cyclization that has been observed before. Shows the steps in the autocatalyzation of the chromophore.. shows cyclization. Aequorea Victoria Fluorescent Proteins.1 Then the amide group of Gly67 does a nucleophilic attack on the carbonyl carbon of Try66 to cyclize and form the imidazolidin ring. 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.fsu. Top row.Try66. Richard et. Even once it is made.html) chromophore.2 The next step of chromophore formation is dehydration. leaving behind an imidazolin-‐5-‐one intermediate. left to right.edu/articles/probes/jellyfishfps. (Day.1 When oxygen is re-‐administered the GFP gains fluorescence. because after it is formed. where water comes off of Ser65. Bottom row. oxygen will quench its fluorescence.magnet. http://zeiss-‐ campus.1 This is strange that molecular oxygen is needed in the formation of the chromophore.2 It is essential to have Gly as residue 67. left to right.1 The last step is reduction of the alpha-‐beta bond of Try66 by molecular oxygen. and oxidation to produce the mature chromophore. the chromophore needs to be surrounded by the rest of the protein in order to fluoresce.
Green Fluorescent Protein: A perspective. (Remington. The structure of green fluorescent protein is essential for proper protein function. emission peaks at 508nm.fluorescence. keeping the interactions with the chromophore very predictable to preserve the ratio. either neutral (A form) or anionic (B form). When excited at 395nm. favoring the neutral. GFP has a major peak of absorption at 375nm in the UV range. A is the neutral and B is the anionic form of the chromophore. these are always seen in a 6:1 ratio.1 Another physical characteristic of GFP is its fluorescence. and a minor peak at 475nm which is the green visible light that we see. This is because the structure of GFP effectively protects the chromophore from outside influences. The neutral chromophore absorbs at 395nm. 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. 7 . “The naked chromophore is neither rigid nor protected from jostling by solvent molecules” so it cannot yet fluoresce.2 These two peaks in absorbance at 375nm and 479nm are due to the two different states the chromophore can take. Without its specific structure this protein would not be fluorescent or would not nearly as useful as it is as a biological marker. so it needs the protection of the beta barrel. keeping it in the proper conformation to fluoresce. For example. The chromophore is also assisted by other residues to go from neutral to anionic. 2011. By mutating key residues. the beta-‐barrel protects the chromophore. while the anionic form absorbs at Figure 4. The absorbance peaks at 395nm and 475nm and the chromophore structures that cause them.) 475nm. In wild-‐type GFP.
This GFP Chromophore is a strong photoacid as upon the excitation of the chromophore by the photon. one major peak at 396 nm and a minor peak at 475 nm. This overall function of GFP occurs only after a series of events take place. This green light emission is principally and directly the product of the Green Fluorescent Protein chromophore.0 to 1.8 The chromophore is composed of Serine 65. The biological mechanism for the ultimate emission of the green photon in wtGFP (from The GFP Site. it takes on acidic properties. as discussed earlier.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. a calcium ion must activate the Aequorin photoprotein. but can also be forced into its o-‐HBDI conformation at no loss of fluorescence. and Glycine 67 which autocatalyze to form the mature GFP chromophore.conncoll. which in turn will release a photon of blue light with an emission peak at 470 nm that can activate GFP. a series of events that depend on the type of GFP being studied.0.9 This acidic nature of the chromophore will later play a key role in the deprotonation of the chromophore. Since GFP has two excitation peaks. an event 8 . research suggests that there is more than one way to excite the photoprotein and result in the emission of the green light.edu/) emission of the photon with the capability of activating GFP. Tyrosine 66. This occurrence was observed when upon photon absorption the pKa of the chromophore changed from 8. The chromophore is naturally found in the p-‐HBDI conformation. For example. in vitro studies utilize UV light for the Figure 5. while in wtGFP. http://gfp.
ibch.that ultimately leads to the fluorescence emitted from the mature chromophore. the chromophore must naturally hydrogen bond with other key residues. http://www. This blue photon excites the GFP chromophore allowing for wild-‐type GFP to emit its green fluorescence. to Serine 205. 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. The network of hydrogen bonding in which the chromophore participates (from The Institute of Bioorganic Chemistry of the Russian Academy of Sciences. The chain of hydrogen bonds that specifically participate in the excited state proton transfer is the relay from the chromophore’s Tyrosine 66 residue to a water molecule (often numbered residue 285). the GFP chromophore is interconnected to many other residues both on the α-‐helix and a part of the β-‐barrel. retaining the integrity of the chromophore by ensuring it remains planar.6 Excited State Proton Transfer. and most importantly. Upon the extraction of GFP from the Aequorea victoria. The network of hydrogen bonding both stabilizes the chromophore.ru/en/structure/groups/xray) activated by the absorption of a photon. Figure 7 shows the absorbance of the photon of light (denoted hv) by the A state of the chromophore. However. In fact. called Aequorin. is Figure 6. scientists found that the neighboring photoprotein. it facilitates the chain of proton transfers from the chromophore to surrounding residues. or ESPT for short. As depicted and described 9 . and finally to Glutamic Acid 222. was responsible for releasing this blue colored photon when activated to do so by a Calcium ion. in order to become deprotonated.
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. 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. 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. this state does exist and is a 10 . and once the water molecule is protonated. the photoacidic properties of the chromophore result in an acidic chromophore once it absorbs the blue emission.10 The deprotonation of the chromophore and the ultimate protonation of the Glutamic Acid 222 residue produce the I*-‐state of the chromophore. absorbance of the photon and the excitation of the chromophore causes Tyrosine 66 to become deprotonated. or the excited intermediate state. James. This is the rate-‐determining step.earlier. This is the state of the chromophore that releases the green fluorescence. Excited State Proton Transfer Mechanism (from Remington. Due to its acidic properties. Green Fluorescent Protein: A Perspective. 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.6 The other equilibrated state that the chromophore can take on is called the B-‐state. transferring its proton to the neighboring hydrogen-‐bonding partner. 2011) 222 residue all occur within picoseconds. However. S. the water molecule. or the anionic state.
resulting in a more efficient fluorescence. this state may also be excited when it absorbs a photon at 475 nm and changes into the excited B state. Over the past decade. In the B state. otherwise the mutation will be detrimental to the protein and the green fluorescence properties of GFP will disappear. Martin Chalfie. there is no excited state proton transfer that will result in the fluorescence of the green photon.simple conformation change of the Glutamic Acid 222 residue from the I state. However. a prime example of this increase was the awarding of the 2008 Nobel Prize in Chemistry to Roger Tsien. the natural state of GFP was not good enough for many scientists. which also emits the green fluorescing photon. even greater than the fluorescence that resulted from the 396 excitation peak. Whereas I*-‐state is indirectly excited when the A-‐state becomes excited. Instead. Roger Tsien. resulting in the emission of the green fluorescence. They tested many 11 .11 Just recently. interest in GFP has spiked. and Andrew Cubitt set out to improve the fluorescence resulting from a 475 nm excitation. but was relatively low in amplitude. upon the observation that the 475 nm excitation fluorescence had a good photostability. because the syn conformation prevents the hydrogen bond from being formed between the Glutamic Acid 222 residue and the Serine 205 residue. the I state being the anti conformer and the B state being the syn conformer. much of the research done regarding Green Fluorescent Protein has been observing the results of mutations on the structure and function of GFP. the B state excites and fluoresces directly. and Osamu Shimomura for the discovery and development of GFP. B*-‐ state. One important thing to remember about any protein however is that structure determines function and because of this. In 1995. One of the most important and biggest discoveries in the field of mutations to GFP was the substitution of Threonine for Serine 65. mutations must result in a conservation in packing and folding. Roger Heim.
al. In Figure 9. that in its own way can result in the fluorescence of the protein. Steven R. and Threonine to see if. Excitation (dashed lines) and emission (solid lines) spectra of wt GFP (gray lines) and GFP-‐S65T (black lines). there are two clear excitation peaks.different amino acids such as Alanine. This would be expected due to both forms of the chromophore can take on. (from Kain... et. one oddity that the figure presents shows up in the representation of the excitation peaks. 1996) the mutated S65T is significantly higher than the emission peak of the wild type GFP. Cysteine. Transient Expression of a Mutated GFP cDNA Leads to Improved Fluorescence in Plant Protoplasts. further investigation found that the alteration of Serine 65 to Threonine restricts the excited state 12 . Fluorescence observed in GFP extracted from cDNA (left). In 1997. Fluorescence observed in S65C mutation (right) (from Reichel et. However. but they decided that the most practical mutation was the substitution of Threonine for Serine 65 (S65T). like Serine. one at around 396 nm and the Figure 9.12 Although the S65T mutant results in stronger fluorescence. in the S65T mutant. because the Threonine mutant resulted in the longest wavelength of excitation and the highest relative amplitude upon excitation by an external photon. al. the relative amplitude of Figure 8. Expression and Detection of Green Fluorescent Protein (GFP). In wild type GFP. excitation of the mutated chromophore would produce a vinyl side chain and either H2O or H2S (in the case of Cysteine). Leucine. 1997) other at around 475 nm. To their surprise. each of these amino acid mutations procured the desired results. either A or B state. there is only one excitation peak around the 475 nm mark instead of the normal two peaks.
proton transfer from the chromophore to Glutamic Acid 222 and instead. a group of scientists in 2008 became determined to find a way to recover this loss of fluorescence. This mutant is referred to as the E222Q mutant. B) Hydrogen bonds in which Tyrosine 66 and water participate (from Shinobu. This mutation in both the case of the S65T mutant and the E222Q mutant recovered its absorbance at the 396 nm wavelength. through water and Serine 205 to Glutamic Acid 222. Because Glutamine doesn’t possess the same acidic properties that Glutamic Acid has. Al et. A) Overall series of hydrogen bonds connecting the chromophore to E222 residue. al. with Glutamine.11 Another mutation that is similar in nature to the S65T mutation is the replacement of Glutamic Acid 222. straight to the Aspartic Acid 148 residue. Visualizing Proton Antenna in a High-Resolution Fluorescent Structure. Glutamine can’t participate in excited state proton transfer. This discovery proved that there is more than one pathway for 13 . solely producing green emission through the excitation of the B-‐state. However.. the proton wasn’t transferred Figure 10. thus also eliminating fluorescence through the excitation of the A-‐state.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. representing a recovery of excited state proton transfer. but rather. 2011) normally. the final protonated residue in the series of excited state proton transfers. fluoresces solely through the excitation of the B-‐state. Their answer? Replace Histidine 148 with Aspartic Acid.
14 14 . Threonine 203 actually rotates and repositions itself to hydrogen bond with the anionic-‐B-‐state. By swapping out Histidine for Threonine 203. It was determined that the reason for the loss of absorption through the B-‐state is that upon photoactivation of the wild-‐type GFP. further stabilizing the formation of the B-‐state chromophore. Note the missing peak for PA-‐GFP at the 475 nm mark). the A-‐state. 2008) for this mutant and the peak at 396 nm is amplified.11 Another important mutation that was discovered was the substitution of Histidine for Threonine 203. just like how for the S65T mutant. J. Absorption and Fluorescence emission spectra of native PA-‐GFP. Structure and Mechanism of the Photoactivatable Green Fluorescent Protein. This means that the photon excitation peak at 475 nm for wild type GFP doesn’t exist Figure 11. the T203H mutant promotes the excitation of the A-‐state chromophore and instead inhibits fluorescence through the B-‐state of the chromophore. resulting in what is called the photoactivatable GFP or PA-‐GFP. the 475 nm excitation peak was amplified and the 396 nm excitation peak was gone altogether (See Figure 11. al. and wild-‐type GFP (from Henderson. this rotation is not an option and Histidine 203 remains unaltered upon photoactivation in PA-‐ GFP. While the S65T and E222Q mutants halt the excitation of the chromophore’s A-‐state at 396 nm. photoactivated GFP..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. Nathan et.
It is this 15 . the study showed that instead of having to rely on outside residues for proton transfer in either the β-‐barrel or the α−helix and chromophore maturation. as seen in Figure 12. Comprehensive Studies on an Overall Proton Transfer Cycle of the ortho-Green Fluorescent Protein Chromophore. Structure of p-HBDI and o-HBDI (from Hsieh. Structurally. 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. Cheng-‐Chih et. when the o-HBDI conformer was excited in much the same way as the p-HBDI. Cheng-‐Chih et. one of the bonds being made by a hydrogen bond between the O-‐R group and the nitrogen of the imidazoline ring. 2011) mature as o-HBDI. Mechanism of o-HBDI (from Hsieh. a slightly different conformation. Published in the Journal of the American Chemical Society on February 16. After further studying of the exact mechanism of the o-HBDI conformer of the chromophore. However. al.. scientists forced the naturally forming p-HBDI (the GFP chromophore) to 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. ortho-‐HBDI is very similar. al. is Figure 13. The only difference is the intrinsic seven-‐membered ring. it also resulted in fluorescence. shown in Figure 13.. 2011) excited. Comprehensive Studies on an Overall Proton Transfer Cycle of the ortho-Green Fluorescent Protein Chromophore. 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. 2011. naturally occurring GFP chromophore.
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. In this study. and 50 vol % [bmim]Cl (green) (from Heller. one with 25% and the other with 50% of the ionic liquid by volume. 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. William et.4) (black). Another effect of the ionic liquid was that the protein appeared less compact in the ionic liquid than when it was dissolved in water. both the bottom curves belonging to the GFP dissolved in the ionic 16 Figure 14. GFP was placed in two different assays of the ionic liquid 1-‐butyl-‐3-‐methylimidazoline chloride. 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.tautomer intermediate that allows for the fluorescence to occur when the chromophore is in the o-HBDI conformer. it is the intermediate state. water (red). 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. Denaturation Curves for GFP measured in 10 mM Tris·HCl (pH 7. which implies that there was slight unfolding in the structure of the protein. 25 vol % [bmim]Cl (blue). 2010) .15 For many years.. However. Characterization of the Influence of the Ionic Liquid 1- Butyl-3-methylimidazoline Chloride on the Structure and Thermal Stability of Green Fluorescent Protein. that fluoresces.al. much like the how in the p-HBDI conformer. I*-‐state.
has just recently garnered attention for its phenomenal properties and the role it plays in cell imaging. has been heading towards the observation and understanding of the effects of stressors on the structure and function of GFP. However. 17 . 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. environmental effects. In both cases. From this information. as evidenced by the numerous studies outlined. being a relatively new molecule. Green Fluorescent Protein remains an integral aspect of science today.16 Green Fluorescent Protein. Also just recently. and through uses such as “Brainbow”. whether it be mutations or outside.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. interfere with the crystal structure of the GFP protein and hinder its function greatly. Green Fluorescent Protein: A Perspective. like in the study of mutating Histidine 148 to Aspartic Acid. and Yellow fluorescing proteins. James. S. 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. GFP continues to change the face of biological imaging.solutions. it was concluded that the ionic liquid the team had chosen to test. 2011) common derivatives of GFP being the Red. Researchers have also been studying the ability for GFP to recover its fluorescence once restricted. 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. Research in GFP. Orange. if not all ionic liquids.
Y. Tu. Feb 11. 6. Rev. Nature Biotechnology. Y. Laren et. et. 5. al. R. 1994. Cubit.3: 1932-‐ 1947 18 . Journal of American Chemical Society 2007. al. Annu..Works Cited 1. Excited-‐State Intramolecular Proton Transfer via a Seven-‐Membered-‐Ring Hydrogen-‐Bonding System. 203. G. Understanding. M.20: 1509–1519 7. 263. Crystal structure of the Aequorea victoria green fluorescent protein. Cubitt. 35:19-‐27 10.. G.. Kew-‐Yu Chen et. Ormo.Moss. Med Sci (Paris) 2007.. L. 4.W. Tolbert.Euskirchen. al.. Mats. The molecular structure of Green Fluorescent Protein.Prasher... Tsien. Tsien. Excited-‐State Proton Transfer: From Constrained Systems to “Super” Photoacids to Superfast Proton Transfer. Green fluorescent protein as a marker for gene expression.. Chalfie. Livet J.. 1996.Ward.B et.1996. 5148.. Trends in Biochemical Sciences. The Green Fluorescent Protein. The brain in color: transgenic “Brainbow” mice for visualizing neuronal circuits.C. Gross. Jung. Sep 6.Jr.20 (11): 448-‐455 2.. pp. PNAS 2002. 1246-‐1251. and 222.Y. R. Yang.9: 2864-‐2865 12. W. Biochem 1998. Ortho Green Fluorescence Protein Synthetic Chromophore. Karen. Markus A. Proton Shuttle in Green Fluorescent Protein Studied by Dynamic Simulations. S. Phillips. J. al. A. 14 (10).G..129:4534-‐ 4535 9. al. al. Science. Kallio. Biophysical Journal 2005. Remington.. 5280. Improved Green Fluorescence. D.. Green Fluorescent Protein: A Perspective. Gregor et. Science. Protein Science 2011. 1995.23:1173–1176. [in:French] 8. 273.N. Andrew B. F. Observation of Excited-‐State Proton Transfer in Green Fluorescent Protein using Ultrafast Vibrational Spectroscopy. Tsien. Accounts of Chemical Research 2002. improving and using green fluorescent proteins. Lill.Remington.373: 663-‐664 13. S. Deborah et.67: 509–544 3.J. Stoner-‐Ma. Nature 1995. Roger et.5: 2778-‐2781 11. Larry A. Journal of American Chemical Society 2004. The Photophysics of Green Fluorescent Protein: Influence of the Key Amino Acids at Positions 65. al.
J. Heller.. Henderson. Journal of American Chemical Society 2010.131: 4176-‐4177 15. Journal of American Chemical Society 2011.al.14.133: 2932-‐2943 16. Nathan et. Cheng-‐Chih et. William et.. Journal of American Chemical Society 2009. Structure and Mechanism of the Photoactivatable Green Fluorescent Protein.114: 13866-‐13871 19 . al.. Hsieh. Characterization of the Influence of the Ionic Liquid 1-‐Butyl-‐3-‐ methylimidazolium Chloride on the Structure and Thermal Stability of Green Fluorescent Protein. Comprehensive Studies on an Overall Proton Trasnfer Cycle of the ortho-‐Green Fluorescent Protein Chromophore. al.
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