Sandbox 719: Difference between revisions

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The DsRed fluorescent protein 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). 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 beta barrel with a coaxial helix.

The DsRed fluorescent protein 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 beta barrel with a coaxial helix.

==== 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.]]

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 accessible surface area of the isolated monomer ~~(21)~~ is ~~􏰊10~~,230 Å2, and that of the tetramer is 31,420 Å2, so roughly 25% of the monomer surface is not solvent accessible in the tetramer.

The accessible surface area of the isolated monomer <ref>The interpretation of protein structures: estimation of static accessibility. Lee B, Richards FM. PubMed : [pubmed?term=The%20interpretation%20of%20protein%20structures%3A%20estimation%20of%20static%20accessibility here]</ref> is around 10,230 Å², and that of the tetramer is 31,420 Å2, so roughly 25% of the monomer surface is not solvent accessible in the tetramer.

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.

A unique feature of the DsRed tetramer is the carboxy termini, which in avGFP are flexible [only residues 2–229 are visible of the 238 in the mature protein ~~(7)~~]. In DsRed, the carboxy terminus of the A monomer embraces the C monomer and vice versa, forming a ‘‘clasp’’ about a local 2-fold.

A unique feature of the DsRed tetramer is the carboxy termini, which in avGFP are flexible [only residues 2–229 are visible of the 238 in the mature protein]<ref>Crystal structure of the Aequorea victoria green fluorescent protein. Ormö M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ. . PubMed : [http://www.ncbi.nlm.nih.gov/pubmed/8703075 8703075]</ref>. In DsRed, the carboxy terminus of the A monomer embraces the C monomer and vice versa, forming a ‘‘clasp’’ about a local 2-fold.

Although the carboxyl-terminal carboxylates are partially exposed to solvent, the intimate association of Leu-225 with another protomer in the tetramer will likely result in nonfunctional carboxyl-terminal fusion constructions with DsRed. Much of the AC interface involves the bulge region of the fold (which contacts the chromophore), suggesting that tetramer formation may be important for correct folding and/or proper establishment of the chromophore environment

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=== 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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@@ Line 19: / Line 19: @@
 <Structure load='1G7K' size='370' frame='true' align='right' scene='Insert optional scene name here' />
-The DsRed fluorescent protein 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). 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 beta barrel with a coaxial helix.
+The DsRed fluorescent protein 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 beta barrel with a coaxial helix.
 ==== 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.]]
 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 accessible surface area of the isolated monomer (21) is 􏰊10,230 Å2, and that of the tetramer is 31,420 Å2, so roughly 25% of the monomer surface is not solvent accessible in the tetramer.
+The accessible surface area of the isolated monomer <ref>The interpretation of protein structures: estimation of static accessibility. Lee B, Richards FM. PubMed : [pubmed?term=The%20interpretation%20of%20protein%20structures%3A%20estimation%20of%20static%20accessibility here]</ref> is around 10,230 Å², and that of the tetramer is 31,420 Å2, so roughly 25% of the monomer surface is not solvent accessible in the tetramer.
 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.
-A unique feature of the DsRed tetramer is the carboxy termini, which in avGFP are flexible [only residues 2–229 are visible of the 238 in the mature protein (7)]. In DsRed, the carboxy terminus of the A monomer embraces the C monomer and vice versa, forming a ‘‘clasp’’ about a local 2-fold.
+A unique feature of the DsRed tetramer is the carboxy termini, which in avGFP are flexible [only residues 2–229 are visible of the 238 in the mature protein]<ref>Crystal structure of the Aequorea victoria green fluorescent protein. Ormö M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ. . PubMed : [http://www.ncbi.nlm.nih.gov/pubmed/8703075 8703075]</ref>. In DsRed, the carboxy terminus of the A monomer embraces the C monomer and vice versa, forming a ‘‘clasp’’ about a local 2-fold.
 Although the carboxyl-terminal carboxylates are partially exposed to solvent, the intimate association of Leu-225 with another protomer in the tetramer will likely result in nonfunctional carboxyl-terminal fusion constructions with DsRed. Much of the AC interface involves the bulge region of the fold (which contacts the chromophore), suggesting that tetramer formation may be important for correct folding and/or proper establishment of the chromophore environment
@@ Line 43: / Line 43: @@
 === 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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