Sandbox 719: Difference between revisions
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The last notable interaction is the hydrogen bonding between Gln-213 and Asn-42, wich is probably important for the red-emitting fluorescence of the DsRed. | The last notable interaction is the hydrogen bonding between Gln-213 and Asn-42, wich is probably important for the red-emitting fluorescence of the DsRed. | ||
The four chromophore in the tetramer form a rectangular array of 27-34 , suggesting that it exist a possible energy transfer between different chromophores. This could be important when we know the maturation of each chromophore have an efficient rate close to only 50 %. | The four chromophore in the tetramer form a rectangular array of 27-34 , suggesting that it exist a possible energy transfer between different chromophores. This could be important when we know the maturation of each chromophore have an efficient rate close to only 50 %. | ||
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=== | ===Analisys and comparison between DsRed and GFP chromophore=== | ||
[[Image:ABC.png |400px|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|]] | |||
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. | |||
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===Mutation studies=== | |||
Several studies were lead on DsRed, by random and directed mutagenesis. These studies allowed to identify the influence of a certain number of amino acids on characteristics or on the 3D structur of the protein, and to understand the mechanism of the fluorophore. | Several studies were lead on DsRed, by random and directed mutagenesis. These studies allowed to identify the influence of a certain number of amino acids on characteristics or on the 3D structur of the protein, and to understand the mechanism of the fluorophore. | ||
The replacement of Lys-70 by a Met (mutation K70M) lead to a green fluorescent protein, very close to GFP. The reason of this modification is that Lys residue interacts directly with the chromophore. In fact Lys-70 is certainly a charged residue and should interacts via a water molecule with the residues Glu-215 and Glu-148 of the chromophore. The replacement by the neutral residue Met make this interaction impossible. Mutation of Asn-42 (which should interacts with Gln-66) can be understood by the same way, and lead also to a green molecule. | The replacement of Lys-70 by a Met (mutation K70M) lead to a green fluorescent protein, very close to GFP. The reason of this modification is that Lys residue interacts directly with the chromophore. In fact Lys-70 is certainly a charged residue and should interacts via a water molecule with the residues Glu-215 and Glu-148 of the chromophore. The replacement by the neutral residue Met make this interaction impossible. Mutation of Asn-42 (which should interacts with Gln-66) can be understood by the same way, and lead also to a green molecule. | ||
An other interesting mutation is the replacement of Lys-83. The effect of this mutation depend of the amino will replace the Lys. Actually, K83R and K83N mutations results in green fluorescent protein, but K83M substitution lead to latge redshift of absorbtion and emission maxima. In fact, substition with an Arg will increased the chain size, and so inhibite the maturation, but substitution with a Met (wich had mainly the same size than Lys, but different charge) change only absorbtion and emission behavior. | An other interesting mutation is the replacement of Lys-83. The effect of this mutation depend of the amino will replace the Lys. Actually, K83R and K83N mutations results in green fluorescent protein, but K83M substitution lead to latge redshift of absorbtion and emission maxima. In fact, substition with an Arg will increased the chain size, and so inhibite the maturation, but substitution with a Met (wich had mainly the same size than Lys, but different charge) change only absorbtion and emission behavior. | ||
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