Green Fluorescent Protein: Difference between revisions
From Proteopedia
Jump to navigationJump to search
No edit summary |
mNo edit summary |
||
| Line 1: | Line 1: | ||
==Background== | ==Background== | ||
Osamu Shimomura, Martin Chalfie and Roger Y. Tsien shared [http://nobelprize.org/nobel_prizes/chemistry/laureates/2008/ the 2008 Nobel Prize in Chemistry] for their for the discovery and development of the [http://en.wikipedia.org/wiki/Green_Fluorescent_Protein green fluorescent protein], GFP.<br> | Osamu Shimomura, Martin Chalfie and Roger Y. Tsien shared [http://nobelprize.org/nobel_prizes/chemistry/laureates/2008/ the 2008 Nobel Prize in Chemistry] for their for the discovery and development of the [http://en.wikipedia.org/wiki/Green_Fluorescent_Protein green fluorescent protein], GFP.<br> | ||
| Line 42: | Line 40: | ||
===Primary & Secondary Structure=== | ===Primary & Secondary Structure=== | ||
{{STRUCTURE_1ema | PDB=1ema | scene name='Green_Fluorescent_Protein/Initial/1' }} | {{STRUCTURE_1ema | PDB=1ema | scene name='Green_Fluorescent_Protein/Initial/1' }}Green fluorescent protein (<scene name='Green_Fluorescent_Protein/Initial/1'>default scene</scene>) is a 21 kDa protein consisting of 238 residues strung together to form a | ||
Green fluorescent protein (<scene name='Green_Fluorescent_Protein/Initial/1'>default scene</scene>) is a 21 kDa protein consisting of 238 residues strung together to form a | |||
<scene name='Green_Fluorescent_Protein/Secondary_structure/1'>secondary structure</scene> of five α-helices and one eleven-stranded β-pleated sheet,<ref name="PDBsum" /> where each strand contains nine to thirteen residues each.<ref name="Ormo" /> (To view the primary and secondary structure of GFP, go to [[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1ema&template=protein.html&r=wiring&l=1&chain=A www.ebi.aci.uk]].) These β-strands display an almost “seamless symmetry” in which only two of the strands vary in structural content.<ref name="Phillips">Phillips GN Jr. 1997. Structure and dynamics of green fluorescent protein. Curr Opin Struct Biol. 7(6):821-827. DOI 10.1016/S0959-440X(97)80153-4.</ref> This β-sheet conforms itself through regular hydrogen bonding into a β-barrel.<ref name="Yang" /> In GFP, the structure is so regular that <scene name='Green_Fluorescent_Protein/Water_stripes/1'>"stripes"</scene> of water molecules (red) can be seen following the structure of the barrel.<ref name="Phillips" /> Together with the α-helices at either end of the molecule, a nearly perfect cylinder is produced, 42Å long and 24Å in diameter,<ref name="Ormo" /> creating what is referred to as a “β-can” formation.<ref name="Phillips" /> The short helical segments at either end of the cylinder form “caps” to further protect the interior of the β-barrel.<ref name="Phillips" /> Overall stability is maintained by this β-can structure, helping to resist unfolding from heat and other denaturants.<ref name="Yang" /> | <scene name='Green_Fluorescent_Protein/Secondary_structure/1'>secondary structure</scene> of five α-helices and one eleven-stranded β-pleated sheet,<ref name="PDBsum" /> where each strand contains nine to thirteen residues each.<ref name="Ormo" /> (To view the primary and secondary structure of GFP, go to [[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1ema&template=protein.html&r=wiring&l=1&chain=A www.ebi.aci.uk]].) These β-strands display an almost “seamless symmetry” in which only two of the strands vary in structural content.<ref name="Phillips">Phillips GN Jr. 1997. Structure and dynamics of green fluorescent protein. Curr Opin Struct Biol. 7(6):821-827. DOI 10.1016/S0959-440X(97)80153-4.</ref> This β-sheet conforms itself through regular hydrogen bonding into a β-barrel.<ref name="Yang" /> In GFP, the structure is so regular that <scene name='Green_Fluorescent_Protein/Water_stripes/1'>"stripes"</scene> of water molecules (red) can be seen following the structure of the barrel.<ref name="Phillips" /> Together with the α-helices at either end of the molecule, a nearly perfect cylinder is produced, 42Å long and 24Å in diameter,<ref name="Ormo" /> creating what is referred to as a “β-can” formation.<ref name="Phillips" /> The short helical segments at either end of the cylinder form “caps” to further protect the interior of the β-barrel.<ref name="Phillips" /> Overall stability is maintained by this β-can structure, helping to resist unfolding from heat and other denaturants.<ref name="Yang" /> | ||
| Line 68: | Line 65: | ||
This quandary led Phillips to study the acid/base chemistry catalyzing the initial cyclization of the chromophore. He found that Arg<sup>96</sup> actually acts as a | This quandary led Phillips to study the acid/base chemistry catalyzing the initial cyclization of the chromophore. He found that Arg<sup>96</sup> actually acts as a | ||
<scene name='Green_Fluorescent_Protein/Arg96/1'>base</scene> by withdrawing electrons through hydrogen bonding with the carbonyl oxygen of Ser<sup>65</sup> to activate the carbonyl carbon for nucleophilic attack by the amide nitrogen of Gly<sup>67</sup>. This mechanism was further supported by ab initio calculations, as well as database searches of similar compounds and protein sequences. Through acid/base chemistry, the chromophore is stabilized by resonance.<ref name="Phillips" /> | <scene name='Green_Fluorescent_Protein/Arg96/1' target='3' >base</scene> by withdrawing electrons through hydrogen bonding with the carbonyl oxygen of Ser<sup>65</sup> to activate the carbonyl carbon for nucleophilic attack by the amide nitrogen of Gly<sup>67</sup>. This mechanism was further supported by ab initio calculations, as well as database searches of similar compounds and protein sequences. Through acid/base chemistry, the chromophore is stabilized by resonance.<ref name="Phillips" /> | ||
===Mutant Studies=== | ===Mutant Studies=== | ||
<applet load='1ema' size='400' frame='true' align='right' scene='Green_Fluorescent_Protein/Secondary_structure/1' name=' | <applet load='1ema' size='400' frame='true' align='right' scene='Green_Fluorescent_Protein/Secondary_structure/1' name='3'/> | ||
Many mutant green fluorescent proteins have been developed in order to further understand the structure and mechanism of the fluorophore. The first mutagenesis studies simply | Many mutant green fluorescent proteins have been developed in order to further understand the structure and mechanism of the fluorophore. The first mutagenesis studies simply | ||
<scene name='Green_Fluorescent_Protein/Truncated_ends/1'>truncated the ends</scene> of the amino acid sequence (<scene name='Green_Fluorescent_Protein/Secondary_structure/1'>see without truncated ends</scene>. NOTE: The structure represented here is already truncated at the carbonyl terminus). Shortening the polypeptide by more than seven amino acids from either terminus lead to a total loss of fluorescence, as well as a complete failure to absorb light at the traditional wavelengths. This is most likely due to the structure of the protein. The last seven amino acid residues of the carboxyl terminus are roughly disordered, and thus do not interfere with the overall structure. After seven residues, however, the capping α-helix structure is disrupted, leading to an unstable or unformed chromophore. The <scene name='Green_Fluorescent_Protein/Amino_terminus/1'>amino terminus</scene> is less understood, but the same principle still applies even though the β-barrel does not begin until residue ten or eleven.<ref name="Yang" /> | <scene name='Green_Fluorescent_Protein/Truncated_ends/1'>truncated the ends</scene> of the amino acid sequence (<scene name='Green_Fluorescent_Protein/Secondary_structure/1'>see without truncated ends</scene>. NOTE: The structure represented here is already truncated at the carbonyl terminus). Shortening the polypeptide by more than seven amino acids from either terminus lead to a total loss of fluorescence, as well as a complete failure to absorb light at the traditional wavelengths. This is most likely due to the structure of the protein. The last seven amino acid residues of the carboxyl terminus are roughly disordered, and thus do not interfere with the overall structure. After seven residues, however, the capping α-helix structure is disrupted, leading to an unstable or unformed chromophore. The <scene name='Green_Fluorescent_Protein/Amino_terminus/1'>amino terminus</scene> is less understood, but the same principle still applies even though the β-barrel does not begin until residue ten or eleven.<ref name="Yang" /> | ||
