Green Fluorescent Protein: Difference between revisions

The β-barrel provides a highly constrained environment that protects the chromophore from the bulk solvent,<ref name="Haldar" /> nearly creating the atmosphere of a vacuum.<ref name="Lammich" />  This is most likely responsible for the small [http://en.wikipedia.org/wiki/Stokes_shift Stoke’s shift], or the small wavelength difference between excitation and emission.<ref name="Ormo" />   

Findings show that fluorescence will not occur from a naked chromophore, but rather requires the protection of the β-can structure.<ref name="Cubitt" />  However, ''in crystallum'' GFP will exhibit a nearly identical fluorescence spectrum and lifetime when compared with aqueous GFP.  These two elements point to a fluorescence that is not inherent to the isolated fluorophore,<ref name="Yang" /><ref name="Phillips" /> but rather from the auto-catalytic cyclization of the polypeptide sequence Ser⁶⁵Tyr⁶⁶Gly⁶⁷ and subsequent oxidation of Tyr⁶⁶.<ref name="Phillips" />  However, this sequence is found in many proteins - why does GFP fluoresce?  According to Phillips (1997), fluorophore formation is due to the close proximity of the backbone atoms between Ser⁶⁵. and Gly⁶⁷ gained through a lack of sterical hindrance by the hydrogen atom side chain of glycine.  In fact, no functional fluorescent proteins have been found in which any other amino acid other than glycine was found at position 67.  Even so, there are still proteins that have this specific sequence, therefore, there must be another inherent property to GFP that is still left misunderstood.<ref name="Phillips" />   

In high protein concentrations, GFP has been found to dimerize under the influence of high ionic strength between the two monomers.  In ''Aequorea victoria'', the aequorin is able to bind to the <scene name='Green_Fluorescent_Protein/1gfl/1'>dimer</scene> ([[1gfl]]), but not the monomer.  Therefore, dimerization is a very important structural feature in terms of its function, as it also assists the GFP to absorb energy at the excitation wavelength of aequorin even though GFP has only a “modest” extinction coefficient.  As a result, dimers, and often even higher <scene name='Green_Fluorescent_Protein/1w7s/1'>multimers</scene> ([[1w7s]]), are predominant protein populations within the jellyfish.<ref name="Cubitt" />  

==Use in the Laboratory==

*Sapphire: excitation spectra at 495 nm has been suppressed while the 395 nm  spectra is retained; still emits light at 508 nm.

*<scene name='Green_Fluorescent_Protein/Yfp_chromophore/1'>YFP</scene> ([[1yfp]]): red-shifted GFP that has replaced <scene name='Green_Fluorescent_Protein/Yfp_residue_203/1'>residue 203</scene> with an aromatic amino acid.

====Förster Distances (nm) for Energy Transfer====