Sandbox 719: Difference between revisions
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<Structure load='1G7K' size='320' frame='true' align='left' '3D structure of the DsRed, a red fluorescent protein from coral (1G7K) ''Cristalized withe a resolution of 2.0 Å'''/> | <Structure load='1G7K' size='320' frame='true' align='left' '3D structure of the DsRed, a red fluorescent protein from coral (1G7K) ''Cristalized withe a resolution of 2.0 Å'''/> | ||
In this article, we want to present you a red | In this article, we want to present you a red Fluorescent Protein (FP) recently cloned from a corallimorpharian of the ''Discosoma'' genus. The PDB number of this molecule is 1G7K <ref>http://www.rcsb.org/pdb/explore/explore.do?structureId=1G7K</ref>. The publications related is called "Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-Å resolution" <ref>Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-A resolution. Yarbrough D, Wachter RM, Kallio K, Matz MV, Remington SJ. PubMed : [http://www.ncbi.nlm.nih.gov/pubmed/11209050 11209050]. | ||
PubMedCentral : [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC14609/ PMC14609] DOI : [http://www.pnas.org/content/98/2/462 10.1073]</ref> | PubMedCentral : [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC14609/ PMC14609] DOI : [http://www.pnas.org/content/98/2/462 10.1073]</ref> | ||
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=== Introduction === | === Introduction === | ||
Since the discovery of | Since the discovery of FP with the Green Fluorescents Protein (GFP) in the ''Aequorea victoria'' algae, other type of FP was discovered with differents colors such as cyan, yellow and red. All those proteins contribute to the natural coloration of their host and also permit the multicolor tagging experiments. | ||
The crystal structure of DsRed | The crystal structure of DsRed, has been determined at 2.0-Å resolution by multiple-wavelength anomalous dispersion and crystallographic refinement. In this article we want to present you the structure of the protein but also the function, use and a small comparison between de GFP and the DsRed. | ||
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====Secondary Structure==== | ====Secondary Structure==== | ||
<Structure load='1G7K' size=' | <Structure load='1G7K' size='510' frame='true' align='right' scene='Insert optional scene name here' /> | ||
The DsRed | The DsRed is a 28kDa polypeptide that emits a red color after an excitation because of the presents of a chromophore composed by 3 amino acids –Gln-Tyr-Gly- (residues 66-68)<ref>Fluorescent proteins from nonbioluminescent Anthozoa species. Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA.. PubMed : [http://www.ncbi.nlm.nih.gov/pubmed/10504696 10504696]</ref>. The fold of our protein is very closed to the most common fluorescent protein, the ''Aequorea victoria'' Green Fluorescent Protein (avGFP). The primary sequence of those two proteins is 23% similar but in the immediate vicinity of the chromophore the primary structure is strictly conserved and theses several amino acid are probably essential for the chromophore formation. The DsRed protein is obligatory a tetramer in solution. The monomer is composed by 11-strand <scene name='Sandbox_719/Sheet/1'>beta sheet</scene> with a coaxial <scene name='Sandbox_719/Helix/1'>helix</scene>. | ||
==== The tetramer Interface ==== | ==== The tetramer Interface ==== | ||
[[Image:Tetramer-interface.jpg |200px|left|thumb|Figure 1: Ribbon diagram of the DsRed tetramer, produced by MOLSCRIPT (28). Monomers are labeled with uppercase A–D, and the carboxy termini are labeled with lowercase a– d. The amino termini are obscured in this view. Note the antenna-like array of the chromophores, antiparallel in pairs.]] | [[Image:Tetramer-interface.jpg |200px|left|thumb|Figure 1: Ribbon diagram of the DsRed tetramer, produced by MOLSCRIPT (28). Monomers are labeled with uppercase A–D, and the carboxy termini are labeled with lowercase a– d. The amino termini are obscured in this view. Note the antenna-like array of the chromophores, antiparallel in pairs.]] | ||
The most striking feature of DsRed is that, as shown in Figure 1, it exists as an extremely close-packed tetramer, quite unlike the primarily monomeric avGFP. The tetramer is a square prism with remarkably flat sides and a small elliptical hole directly through the center. The hole is lined with polar residues and salt bridges and localizes a number of solvent molecules. | The most striking feature of DsRed is that, as shown in Figure 1, it exists as an extremely close-packed tetramer, quite unlike the primarily monomeric avGFP. It is a dimer of dimer. The whole tetramer is a square prism with remarkably flat sides and a small elliptical hole directly through the center. The hole is lined with polar residues and salt bridges and localizes a number of solvent molecules. | ||
The accessible surface area of the isolated monomer | The accessible surface area of the isolated monomer is around 10,230 Ų, and that of the tetramer is 31,420 Ų, so roughly 25% of the monomer surface is not solvent accessible in the tetramer<ref>The interpretation of protein structures: estimation of static accessibility. Lee B, Richards FM. PubMed : [http://www.ncbi.nlm.nih.gov/pubmed?term=The%20interpretation%20of%20protein%20structures%3A%20estimation%20of%20static%20accessibility here]</ref>. | ||
The AB interface does not seem to have particularly notable features and consists of hydrophobic interactions between small side chains, although a few hydrogen bonds and salt bridges are also present. | The AB interface does not seem to have particularly notable features and consists of hydrophobic interactions between small side chains, although a few hydrogen bonds and salt bridges are also present. | ||
On the other hand, interactions in the AC (and BD) interface consist largely of salt bridges and hydrogen bonds, many of which are mediated by buried water molecules, as well a surprisingly large number of interactions involving aromatic residues. | On the other hand, interactions in the AC (and BD) interface consist largely of salt bridges and hydrogen bonds, many of which are mediated by buried water molecules, as well a surprisingly large number of interactions involving aromatic residues. | ||
==== The Chromophore environment ==== | ==== The Chromophore environment ==== | ||
<Structure load='1G7K' size=' | <Structure load='1G7K' size='490' frame='true' align='left' scene='Insert optional scene name here' /> | ||
[[Image:Chromophore1G7K.gif |250px|right|thumb|Figure 2: Chromophore of the DsRed protein]] | [[Image:Chromophore1G7K.gif |250px|right|thumb|Figure 2: Chromophore of the DsRed protein]] | ||
Structure of the chromophore was predicted by sequence comparisons. | Structure of the chromophore was predicted by sequence comparisons. The chromophore of the DsRed is localized in a very polar cavity, and results to the autocatalytic cyclization and hydrogenation of the<scene name='Sandbox_719/Chromophore/1'> -Gln66-Tyr67-Gly68- tripeptide</scene>. Environment of DsRed chromophore is much more complicated in DsRd than in GFP, cause to the hydrogen bond and salt bridge network. There are more charges in DsRed than in GFP, due to three lysine (residues 70,83 and 163) and glutamic acid (residue 148) wich interact very closely with the chromophore. These charges are localized in two perpendicular band : the positive one is parallel to the axis of the chromophore, and the negative one perpendicular to this axis. | ||
The Phenolate oxygen of the chromophore forms a hydrogen bond with Ser-146 and a nearly water molecule, and it also forms a charge-charge with the Lys-163 residue. To forms all these interactions, the necessarily always charged. | The Phenolate oxygen of the chromophore forms a hydrogen bond with Ser-146 and a nearly water molecule, and it also forms a charge-charge with the Lys-163 residue. To forms all these interactions, the necessarily always charged. | ||
There is an other important residue in the struture of the chromophore, the residue Glu-215 (equivalent to the residue Glu-222 in GFP). This residue is highly close to the chromophore, and it also interacts with a water molecule localized near GLN-66. But Glu-215 also interacts with the positively charged chain of Lys-70, and it forms a hydrogen with Ser-205 side chain, like in GFP. | There is an other important residue in the struture of the chromophore, the residue Glu-215 (equivalent to the residue Glu-222 in GFP). This residue is highly close to the chromophore, and it also interacts with a water molecule localized near GLN-66. But Glu-215 also interacts with the positively charged chain of Lys-70, and it forms a hydrogen with Ser-205 side chain, like in GFP. | ||
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=== Fluorescence === | === Fluorescence === | ||
The broad excitation and emission bands have maxima at 558 and 583 nm, respectively (with a minor peak at 494 nm and a significant tryptophan peak at 280 nm) for a monomer extinction coefficient and fluorescence quantum yield at 558 nm of approximately 75,000 mol-1/cm-1 and 0.7, respectively | The broad excitation and emission bands have maxima at 558 and 583 nm, respectively (with a minor peak at 494 nm and a significant tryptophan peak at 280 nm) for a monomer extinction coefficient and fluorescence quantum yield at 558 nm of approximately 75,000 mol-1/cm-1 and 0.7, respectively<ref>Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Baird GS, Zacharias DA, Tsien RY. PubMed : [http://www.ncbi.nlm.nih.gov/pubmed?term=Biochemistry%2C%20mutagenesis%2C%20and%20oligomerization%20of%20DsRed%2C%20a%20red%20fluorescent%20protein%20from%20coral. here]</ref>. | ||
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===Analisys and comparison between DsRed and GFP chromophore=== | ===Analisys and comparison between DsRed and GFP chromophore=== | ||
[[Image:ABC.png |290px|left|thumb|| (A) A portion of the experimental multiwave- length anomalous dispersion-phased electron density map at 2.0-Å resolution.(B) Ball-and-stick diagram of the DsRed chromophore and environment. (C) Schematic diagram of the chromophore environment showing salt bridges and/or hydrogen bonds]] | [[Image:ABC.png |290px|left|thumb||Figure 3: (A) A portion of the experimental multiwave- length anomalous dispersion-phased electron density map at 2.0-Å resolution.(B) Ball-and-stick diagram of the DsRed chromophore and environment. (C) Schematic diagram of the chromophore environment showing salt bridges and/or hydrogen bonds]] | ||
These experiments shown the existence of a green fluorescent protein intermediate, very close to GFP, and suggests that there are several steps in the overall reaction (Matz et al.). Studies of Baird et al. shown there are two key conserved amino acids : Gln-66 and Gln-215. When we do the comparison of chromophore of DsRed and GFP, two points seems to be important. First Gln-66 of DsRed had a sp2 hybridization while Thr/Ser-65 (which are at the same place in the molecule) of GFP have a sp3 hybridization. Secondly, the conserved glutamate Glu-215 of DsRed is closer of the chromophore and is bonded to a water molecule close to GlN-66, which becomes oxidized. So this positionning of Gln-66 and Glu-215 should have a big influence in the red fluorescence, and Glu-215 could have two roles : In the formation of green fluorescence intermediate, and, by its environment, is crucial for formation of green or red emitting species. | These experiments shown the existence of a green fluorescent protein intermediate, very close to GFP, and suggests that there are several steps in the overall reaction (Matz et al.). Studies of Baird et al. shown there are two key conserved amino acids : Gln-66 and Gln-215. When we do the comparison of chromophore of DsRed and GFP, two points seems to be important. First Gln-66 of DsRed had a sp2 hybridization while Thr/Ser-65 (which are at the same place in the molecule) of GFP have a sp3 hybridization. Secondly, the conserved glutamate Glu-215 of DsRed is closer of the chromophore and is bonded to a water molecule close to GlN-66, which becomes oxidized. So this positionning of Gln-66 and Glu-215 should have a big influence in the red fluorescence, and Glu-215 could have two roles : In the formation of green fluorescence intermediate, and, by its environment, is crucial for formation of green or red emitting species. | ||
Matz et al. Suggested that GFP is a “broken” version of an ancestral red fluorescent protein, due to these obsevations. | Matz et al. Suggested that GFP is a “broken” version of an ancestral red fluorescent protein, due to these obsevations. | ||
