The environment of the chromophore is formed by polar amino acids as well as positively and negatively charged ones. Together with some tightly bound water molecules a web of hydrogen and salt bridges is formed. So the phenolic oxygen of the chromophore's benzyl group is hydrogen bridged to three amino acids and further strongly polarized by a lysine with the effect of the chromophore's oxygen being kept in the charged phenolate state. This keeps the chromophore system filled with electric charge. The imidazolone oxygen is bound to the positively charged guanidino group of an arginine . The glutamine sidechain of the chromophore is fixed by an asparagine and a glutamine . In the mature tetrameric protein only half of the chromophores are oxidized in the second step, so really there is a mixture of green and red fluorescent dyes. An arrangement of mixed oxidation states within one tetramer would provide for an extremely efficient energy transfer between green and red centers (just to remind you: the blue light generated by the primary producer aequorin cannot be absorbed by the red fluorescing dye - the green fluorescing system has to be in between). The regular arrangement of the protein's strands into a beta barrel is disturbed at the position of Glu144 . The carbonyl oxygen of this residue is turned inward to form water mediated hydrogen bridges to the chromophore and therefore is not available to form the beta sheet. From this position up to the rim of the barrel there is a cleft which is widened at the rim by a beta bulge. This bulge originates from two adjacent amino acids in one strand to hydrogen bind to the same amino acid in the opposite strand which disturbs the pleated sheet geometry. The cleft is sealed to the inside by the central helix , but a tiny gap remains open (use the mouse to find the peephole to the chromophore!). In the living coral red fluorescent protein is a highly organized tetramer. A spacefill view (with a colour scheme you learned by now: red for helix, light brown for sheet, yellow for one of the four subunits, water blue) shows the very compact structure with only a narrow hole at the center. How does it stick together in just this pattern? There are specific contacts glueing the proteins together: there are amino acids in one subunit forming a lock around amino acids of the other subunit (shown for one interface only). More hydrophobic interactions keep these subunits together . The interface to the other neighbour is formed by hydrophobic interaction and hydrogen bonds . So red fluorescence is enabled by a two step energy transmission from aequorin to a protein complex harboring a cascade of fluorescing dyes in a physical arrangement yielding the highest quantum efficiency possible. Mind this when you obtain some coral souvenir! (by the time you do the coral will be dead of course). Restart this demonstration Literature: D Yarbrough et al, Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-Å resolution, Proc. Natl. Acad. Sci. USA 98 (2001) 462-467 4-03 - Rolf Bergmann
In the mature tetrameric protein only half of the chromophores are oxidized in the second step, so really there is a mixture of green and red fluorescent dyes. An arrangement of mixed oxidation states within one tetramer would provide for an extremely efficient energy transfer between green and red centers (just to remind you: the blue light generated by the primary producer aequorin cannot be absorbed by the red fluorescing dye - the green fluorescing system has to be in between). The regular arrangement of the protein's strands into a beta barrel is disturbed at the position of Glu144 . The carbonyl oxygen of this residue is turned inward to form water mediated hydrogen bridges to the chromophore and therefore is not available to form the beta sheet. From this position up to the rim of the barrel there is a cleft which is widened at the rim by a beta bulge. This bulge originates from two adjacent amino acids in one strand to hydrogen bind to the same amino acid in the opposite strand which disturbs the pleated sheet geometry. The cleft is sealed to the inside by the central helix , but a tiny gap remains open (use the mouse to find the peephole to the chromophore!). In the living coral red fluorescent protein is a highly organized tetramer. A spacefill view (with a colour scheme you learned by now: red for helix, light brown for sheet, yellow for one of the four subunits, water blue) shows the very compact structure with only a narrow hole at the center. How does it stick together in just this pattern? There are specific contacts glueing the proteins together: there are amino acids in one subunit forming a lock around amino acids of the other subunit (shown for one interface only). More hydrophobic interactions keep these subunits together . The interface to the other neighbour is formed by hydrophobic interaction and hydrogen bonds . So red fluorescence is enabled by a two step energy transmission from aequorin to a protein complex harboring a cascade of fluorescing dyes in a physical arrangement yielding the highest quantum efficiency possible. Mind this when you obtain some coral souvenir! (by the time you do the coral will be dead of course). Restart this demonstration Literature: D Yarbrough et al, Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-Å resolution, Proc. Natl. Acad. Sci. USA 98 (2001) 462-467 4-03 - Rolf Bergmann
The regular arrangement of the protein's strands into a beta barrel is disturbed at the position of Glu144 . The carbonyl oxygen of this residue is turned inward to form water mediated hydrogen bridges to the chromophore and therefore is not available to form the beta sheet. From this position up to the rim of the barrel there is a cleft which is widened at the rim by a beta bulge. This bulge originates from two adjacent amino acids in one strand to hydrogen bind to the same amino acid in the opposite strand which disturbs the pleated sheet geometry. The cleft is sealed to the inside by the central helix , but a tiny gap remains open (use the mouse to find the peephole to the chromophore!). In the living coral red fluorescent protein is a highly organized tetramer. A spacefill view (with a colour scheme you learned by now: red for helix, light brown for sheet, yellow for one of the four subunits, water blue) shows the very compact structure with only a narrow hole at the center. How does it stick together in just this pattern? There are specific contacts glueing the proteins together: there are amino acids in one subunit forming a lock around amino acids of the other subunit (shown for one interface only). More hydrophobic interactions keep these subunits together . The interface to the other neighbour is formed by hydrophobic interaction and hydrogen bonds . So red fluorescence is enabled by a two step energy transmission from aequorin to a protein complex harboring a cascade of fluorescing dyes in a physical arrangement yielding the highest quantum efficiency possible. Mind this when you obtain some coral souvenir! (by the time you do the coral will be dead of course). Restart this demonstration Literature: D Yarbrough et al, Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-Å resolution, Proc. Natl. Acad. Sci. USA 98 (2001) 462-467 4-03 - Rolf Bergmann
In the living coral red fluorescent protein is a highly organized tetramer. A spacefill view (with a colour scheme you learned by now: red for helix, light brown for sheet, yellow for one of the four subunits, water blue) shows the very compact structure with only a narrow hole at the center. How does it stick together in just this pattern? There are specific contacts glueing the proteins together: there are amino acids in one subunit forming a lock around amino acids of the other subunit (shown for one interface only). More hydrophobic interactions keep these subunits together . The interface to the other neighbour is formed by hydrophobic interaction and hydrogen bonds . So red fluorescence is enabled by a two step energy transmission from aequorin to a protein complex harboring a cascade of fluorescing dyes in a physical arrangement yielding the highest quantum efficiency possible. Mind this when you obtain some coral souvenir! (by the time you do the coral will be dead of course). Restart this demonstration Literature: D Yarbrough et al, Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-Å resolution, Proc. Natl. Acad. Sci. USA 98 (2001) 462-467 4-03 - Rolf Bergmann
So red fluorescence is enabled by a two step energy transmission from aequorin to a protein complex harboring a cascade of fluorescing dyes in a physical arrangement yielding the highest quantum efficiency possible. Mind this when you obtain some coral souvenir! (by the time you do the coral will be dead of course). Restart this demonstration Literature: D Yarbrough et al, Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-Å resolution, Proc. Natl. Acad. Sci. USA 98 (2001) 462-467 4-03 - Rolf Bergmann
