|
|
| Line 35: |
Line 35: |
| <applet load='1ema' size='500' frame='true' align='right' scene='Green_Fluorescent_Protein/Polar_interactions/2' name='2'/> | | <applet load='1ema' size='500' frame='true' align='right' scene='Green_Fluorescent_Protein/Polar_interactions/2' name='2'/> |
|
| |
|
| As the central α-helix is not located directly in the center of the β-barrel, cavities of differing area exist on either side of the chromophore. The larger cavity, consisting of about 135 Å,<ref name="Ormo" /> does not open out to the bulk solvent, but rather houses <scene name='Green_Fluorescent_Protein/Water_molecules/1' target='2'>four water molecules</scene>.<ref name="Ormo" /><ref name="Van" /> Had this space not been occupied, it would be expected to considerably destabilize the protein as a whole. The hydrogen bonding created by the presence of the water molecules, however, helps to link the buried <scene name='Green_Fluorescent_Protein/Gln69_glu222/1' target='2'>side chains</scene> of Glu<sup>222</sup> and Gln<sup>69</sup> that would otherwise be actively polar.<ref name="Ormo" /> Therefore, the water molecules are extremely important in establishing a hydrogen bonding network about the chromophor.<ref name="Lammich">PMID: 17040991</ref> | | As the central α-helix is not located directly in the center of the β-barrel, cavities of differing area exist on either side of the chromophore. The larger cavity, consisting of about 135 Å,<ref name="Ormo" /> does not open out to the bulk solvent, but rather houses <scene name='Green_Fluorescent_Protein/Water_molecules/1' target='2'>four water molecules</scene>.<ref name="Ormo" /><ref name="Van">van Thor JJ, Sage, JT. 2006. Charge transfer in green fluorescent protein. Photochemical & Photobiological Sciences. 5:597-602. DOI 10.1039/b516525c.</ref> Had this space not been occupied, it would be expected to considerably destabilize the protein as a whole. The hydrogen bonding created by the presence of the water molecules, however, helps to link the buried <scene name='Green_Fluorescent_Protein/Gln69_glu222/1' target='2'>side chains</scene> of Glu<sup>222</sup> and Gln<sup>69</sup> that would otherwise be actively polar.<ref name="Ormo" /> Therefore, the water molecules are extremely important in establishing a hydrogen bonding network about the chromophor.<ref name="Lammich">PMID: 17040991</ref> |
|
| |
|
| The opposite side of the chromophore, however, is within close proximity of several aromatic and polar side chains. Several <scene name='Green_Fluorescent_Protein/Polar_interactions/2'>polar interactions</scene> between the surrounding residues and the chromophore are present including: hydrogen bonds of His<sup>148</sup>, Thr<sup>203</sup>, and Ser<sup>205</sup> with the phenolic hydroxyl of Tyr<sup>66</sup>; Arg<sup>96</sup> and Gln<sup>94</sup> with the carbonyl of the imidazolidinone ring; and hydrogen bonds of Glu<sup>222</sup> with the side chain of Thr<sup>65</sup>. Additional hydrogen bonding in the area around the chromophore helps to stabilize Arg<sup>96</sup> in the protonated form, which suggests the presence of a partial negative charge on the carbonyl oxygen of the imidazolidinone ring in the deprotonated fluorophore.<ref name="Ormo" /> Arg<sup>96</sup> and Gln<sup>94</sup> in turn help to steady the imidazolidone.<ref name="Yang" /> Therefore, it is thought that Arg<sup>96</sup> is essential for the formation of the fluorophore by catalyzing the initial ring closure.<ref name="Ormo" /> Tyr<sup>145</sup> provides a stabilizing | | The opposite side of the chromophore, however, is within close proximity of several aromatic and polar side chains. Several <scene name='Green_Fluorescent_Protein/Polar_interactions/2'>polar interactions</scene> between the surrounding residues and the chromophore are present including: hydrogen bonds of His<sup>148</sup>, Thr<sup>203</sup>, and Ser<sup>205</sup> with the phenolic hydroxyl of Tyr<sup>66</sup>; Arg<sup>96</sup> and Gln<sup>94</sup> with the carbonyl of the imidazolidinone ring; and hydrogen bonds of Glu<sup>222</sup> with the side chain of Thr<sup>65</sup>. Additional hydrogen bonding in the area around the chromophore helps to stabilize Arg<sup>96</sup> in the protonated form, which suggests the presence of a partial negative charge on the carbonyl oxygen of the imidazolidinone ring in the deprotonated fluorophore.<ref name="Ormo" /> Arg<sup>96</sup> and Gln<sup>94</sup> in turn help to steady the imidazolidone.<ref name="Yang" /> Therefore, it is thought that Arg<sup>96</sup> is essential for the formation of the fluorophore by catalyzing the initial ring closure.<ref name="Ormo" /> Tyr<sup>145</sup> provides a stabilizing |
| Line 59: |
Line 59: |
| An interesting mutation discovered by Ormo et al. (1996) was the Thr<sup>65</sup>Tyr<sup>66</sup>Gly<sup>67</sup> mutant, which produces an α-helical conformation in the chromophore opposed to the normal conformation, which is nearly perpendicular to the helical axis, due to its interaction with Arg<sup>96</sup>. This further supports the idea that Arg<sup>96</sup> is an important factor in the structural arrangement required for cyclization, perhaps by promoting the attack of Gly<sup>67</sup> on the carbonyl carbon of Thr<sup>65</sup>.<ref name="Ormo" /> | | An interesting mutation discovered by Ormo et al. (1996) was the Thr<sup>65</sup>Tyr<sup>66</sup>Gly<sup>67</sup> mutant, which produces an α-helical conformation in the chromophore opposed to the normal conformation, which is nearly perpendicular to the helical axis, due to its interaction with Arg<sup>96</sup>. This further supports the idea that Arg<sup>96</sup> is an important factor in the structural arrangement required for cyclization, perhaps by promoting the attack of Gly<sup>67</sup> on the carbonyl carbon of Thr<sup>65</sup>.<ref name="Ormo" /> |
|
| |
|
| In high protein concentrations, GFP has been found to dimerize under the influence of high ionic strength between the two monomers. In ''Aequorea victoria'', the aequorin is able to bind to the <scene name='Green_Fluorescent_Protein/1gfl/1' target='A'>dimer</scene> ([[1gfl]]), but not the monomer. Therefore, dimerization is a very important structural feature in terms of its function, as it also assists the GFP to absorb energy at the excitation wavelength of aequorin even though GFP has only a “modest” extinction coefficient. As a result, dimers, and often even higher <scene name='Green_Fluorescent_Protein/1w7s/1' target='A'>multimers</scene> ([[1w7s]]), are predominant protein populations within the jellyfish.<ref name="Cubitt" /> | | In high protein concentrations, GFP has been found to dimerize under the influence of high ionic strength between the two monomers. In ''Aequorea victoria'', the aequorin is able to bind to the <scene name='Green_Fluorescent_Protein/1gfl/1' target='A'>dimer</scene> ([[1gfl]]), but not the monomer. Therefore, dimerization is a very important structural feature in terms of its function, as it also assists the GFP to absorb energy at the excitation wavelength of aequorin even though GFP has only a “modest” extinction coefficient. As a result, dimers, and often even higher <scene name='Green_Fluorescent_Protein/1w7s/1' target='A'>multimers</scene> ([[1w7s]]), are predominant protein populations within the jellyfish.<ref name="Cubitt">[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TCV-40W0TN7-50&_user=4187488&_coverDate=11%2F30%2F1995&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000062504&_version=1&_urlVersion=0&_userid=4187488&md5=e92730038bb92b1dfbd4af45a0283cce],Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien R. 1995. Understanding, improving, and using green fluorescent protein. Trends in Biochemical Sciences. 20(11): 448-455. DOI 0.1016/S0968-0004(00)89099-4.</ref> |
|
| |
|
| {{Link Toggle FancyCartoonHighQualityView}}. | | {{Link Toggle FancyCartoonHighQualityView}}. |
|
| |
| ==Use in the Laboratory==
| |
|
| |
| Green fluorescent protein has had great success as a marker protein in a variety of biological systems due to its inherent stability, which only adds to its many other desirable characteristics as a marker proteins.<ref name="Haldar" /> GFP is rather resistant to denaturation,<ref name="Yang" /> sustaining structure and function up to 65°C, pH 11, 1% [http://en.wikipedia.org/wiki/Sodium_lauryl_sulfate sodium dodecyl sulphate (SDS)], or 6 M [http://en.wikipedia.org/wiki/Guanidinium_chloride guanidinium chloride]. GFP can also withstand the presence of most proteases for many hours.<ref name="Cubitt">[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TCV-40W0TN7-50&_user=4187488&_coverDate=11%2F30%2F1995&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000062504&_version=1&_urlVersion=0&_userid=4187488&md5=e92730038bb92b1dfbd4af45a0283cce],Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien R. 1995. Understanding, improving, and using green fluorescent protein. Trends in Biochemical Sciences. 20(11): 448-455. DOI 0.1016/S0968-0004(00)89099-4.</ref> Due to this stability, GFP could be applied to numerous other applications such as cell lineage tracing, gene expression reporting, or protein-protein interactions.<ref name="Yang" />
| |
|
| |
| While GFP can be incorporated into most prokaryotic systems, expression in eukaryotic systems may be limited to the cytoplasm and the nucleus, as GFP does not penetrate the nucleolus or vesicular organelles. However, highly specific intracellular localization can still be achieved in eukaryotes,<ref name="Yang" /> which can help to avoid the difficulties associated with adding extrinsic dyes.<ref name="Van">van Thor JJ, Sage, JT. 2006. Charge transfer in green fluorescent protein. Photochemical & Photobiological Sciences. 5:597-602. DOI 10.1039/b516525c.</ref> However, this ability to generate fluorescence within live tissues in the absence of cofactors gives GFP the key for use in biological research. After an in-frame fusion to the protein of interest, the resulting chimeric protein can be expressed in a cellular environment to monitor function or activity<ref name="Pollock">[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TCX-3WRB4G1-5&_user=4187488&_coverDate=02%2F01%2F1999&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000062504&_version=1&_urlVersion=0&_userid=4187488&md5=d50508d087b9a1e500d68a17d2cdb69a], Pollock, BA, and Heim, R. 1999. Using GFP in FRET-based applications. Trends in Cell Biology. 9 (2): 57-60. DOI 10.1016/SO962-8924(98)01434-2.</ref>.
| |
|
| |
| ===Use of GFP as a Fusion Tag===
| |
| One of the most common applications of GFP in research utilizes GFP as a fusion tag to a protein of interest in order to observe the activity of the protein. GFP is fused in frame with the gene encoding the protein of interest, resulting in a chimera that is both functional (hopefully) and fluorescent to be expressed in the organism. This technique has been used successfully in nearly every cell organelle, including the plasma membrane, nucleus, endoplasmic reticulum, Golgi apparatus, secretory vesicles, mitochondria, peroxisomes, vacuoles, and phagosomes. GFP is most commonly fused to either terminal end of the protein gene, although it may be possible to insert it onto a noncritical exterior loop or domain depending on the structure of the protein in question. For example, residues 2-233 of GFP were inserted between the last transmembrane segment and long cytoplasmic tail of a Shaker potassium channel in experiments done by Siegel and Isacoff (1997).<ref name="Tsien" />
| |
|
| |
| ===Use of GFP as an Active Indicator===
| |
| Because the β-can structure protects the chromophore from the surrounding environment, it is difficult to use wild-type GFP as an active indicator of changing conditions. However, environmental indicators have been created by combining various GFP mutants created from random and directed mutagenesis. It is also possible to incorporate phosphorylation sites into the GFP structure such that phosphorylation or dephosphorylation will induce major changes in fluorescence. For example, the Shaker fusion protein mentioned in the section above was the first genetically encoded optical sensor of membrane potential that induced at most a 5% decrease in fluorescence in phosphorylated conditions. However, the most general way to make biochemically sensitive GFPs is to exploit FRET as described in the following sections.<ref name="Tsien" />
| |
|
| |
| ===Use of GFP in FRET===
| |
| [http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer Förster resonance energy transfer] (FRET) is a laboratory technique used to detect the proximity of two biological molecules by the fusion of fluorescent proteins with each of the proteins of interest. When the two fluorophores come within a certain proximity (typcially <100Å) in the proper orientation, the excited fluorophore (donor) emits energy that excites the second longer-wavelength fluorophore (acceptor) suc that it also fluoresces. Results can then be seen by the ratio of the donor and acceptor emission intensities. FRET is noninvasive and is therefore safe for using within live cells<ref name="Pollock" />.
| |
|
| |
| While the wild-type GFP protein has not been particularly useful as a sensor in FRET, several mutants of GFP have been manufactured to create proteins with distinct fluorescence spectra. FRET has been done between two GFP molecules or a single GFP molecule and a secondary fluorophore <ref name="Pollock" />.
| |
|
| |
| ====GFP Mutants Used In FRET====
| |
|
| |
| <applet load='1ema' size='400' frame='true' align='right' scene='Green_Fluorescent_Protein/Chromophore/1' name='5'/>
| |
| (Green links depict the chromophore of each GFP variant. The applet is currently showing the chromophore of <scene name='Green_Fluorescent_Protein/Chromophore/1'>wild-type GFP</scene>.)
| |
|
| |
| *GFP (eGFP): contains a mutation at Ser<sup>65</sup> in which the residue is most commonly replaced by Thr, Ala, or Gly. Lacks the 395 nm excitation peak, but still emits light at 508 nm like wild-type GFP.
| |
| *<scene name='Green_Fluorescent_Protein/Bfp_chromophore/1'>BFP</scene> ([[1bfp]]): blue-shifted GFP that has replaced Tyr<sup>66</sup> with His (Y66H mutation).
| |
| *<scene name='Green_Fluorescent_Protein/Cfp_chromophore/1'>CFP</scene> ([[1oxd]]): contains Y66H mutation with an emission spectra between BFP and eGFP.
| |
| *Sapphire: excitation spectra at 495 nm has been suppressed while the 395 nm spectra is retained; still emits light at 508 nm.
| |
| *<scene name='Green_Fluorescent_Protein/Yfp_chromophore/1'>YFP</scene> ([[1yfp]]): red-shifted GFP that has replaced <scene name='Green_Fluorescent_Protein/Yfp_residue_203/1'>residue 203</scene> with an aromatic amino acid.
| |
|
| |
| {{Link Toggle FancyCartoonHighQualityView}}.
| |
|
| |
| {{Clear}}
| |
| ====Förster Distances (nm) for Energy Transfer====
| |
| {| class="wikitable" border="1" width="100%" height="120%" style="text-align:center"
| |
| |-
| |
| !
| |
| ! colspan="5" align="center"| Acceptor (GFP variant or DsRed)
| |
| |-
| |
| ! Donor
| |
| ! style="background:blue; color:white" | Blue
| |
| ! style="background:cyan; color:black" | Cyan
| |
| ! style="background:green; color:white" | Green
| |
| ! style="background:yellow; color:black" | Yellow
| |
| ! style="background:red; color:white" | Red
| |
| |-
| |
| ! style="background:blue; color:white" | Blue
| |
| | 2.61 ± 0.05
| |
| | 3.77 ± 0.08
| |
| | 4.14 ± 0.08
| |
| | 3.82 ± 0.08
| |
| | 3.17 ± 0.06
| |
| |-
| |
| ! style="background:cyan; color:black" | Cyan
| |
| | -
| |
| | 3.28 ± 0.07
| |
| | 4.82 ± 0.10
| |
| | 4.92 ± 0.10
| |
| | 4.17 ± 0.08
| |
| |-
| |
| ! style="background:green; color:white" | Green
| |
| | -
| |
| | 1.93 ± 0.04
| |
| | 4.65 ± 0.09
| |
| | 5.64 ± 0.11
| |
| | 4.73 ± 0.09
| |
| |-
| |
| ! style="background:yellow; color:black" | Yellow
| |
| | -
| |
| | 1.00 ± 0.02
| |
| | 3.25 ± 0.07
| |
| | 5.11 ± 0.10
| |
| | 4.94 ± 0.10
| |
| |-
| |
| ! style="background:red; color:white" | Red
| |
| | -
| |
| | 1.40 ± 0.03
| |
| | 2.84 ± 0.06
| |
| | 3.14 ± 0.06
| |
| | 3.54 ± 0.07
| |
| |}
| |
| *Data provided by <ref name="Patterson">[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-45FK4Y8-6M&_user=4187488&_coverDate=09%2F10%2F2000&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000062504&_version=1&_urlVersion=0&_userid=4187488&md5=5d93f291367d3192e962e43cc319f6d6], Patterson, GH, Piston, DW, and Barisas, BG. 2000. Förster Distances between Green Fluorescent Protein Pairs. Analytical Biochemistry. 284 (2): 438-440. DOI 10.1006/abio.2000.4708.</ref>
| |
|
| |
| ====Common Pairs of GFP Molecules Used in FRET====
| |
|
| |
| '''BFP-eGFP''': The BFP-eGFP donor-acceptor pair is the most traditionally used pair of GFP molecules used in FRET, but BFP is only weakly fluorescent, creating a limitation for most applications aside from microscopy and flow cytometry.
| |
|
| |
| '''CFP-YFP''': This pair has been used more recently as an alternative to the BFP-eGFP pair because CFP is significantly brighter than BFP, allowing for more accurate ratiometric measurement of donor to acceptor emissions. However, filters must be used carefully while using CFP-YFP in FRET because of the bleeding of the CFP spectra into the YFP spectra. Therefore, proper filters must be set in order to distinguish the fluorescence from donor versus acceptor <ref name="Pollock" />.
| |
|
| |
|
| ==Related Structures== | | ==Related Structures== |
