GFP, Green Fluorescent Protein, its structure was solved in the late 1996. It is said to have a unique soda can. Eleven beta-strands make up the beta-barrel and an alpha-helix runs through the center. The LIGHT IN THE CAN which is located in the middle of the beta barrel is called occasionally and is referred to as the microscopic chromophore. The remarkable cylindrical fold of the protein seems ideally suited for the function of the microscopic protein. Studies and research states that strands of -sheet are tightly fitted to each other like staves in a barrel, and form a regular pattern of hydrogen bonds. Together with the short -helices and loops on the ends, the ‘can’ structure forms a single compact domain and does not have obvious clefts for easy access of diffusable ligands to the microscopic fluorophore. This fold, taken with the observation of a microscope that the microscopic fluorophore is near the geometric center of the molecule explains the observed protection of the microscopic fluorophore from collisional quenching by oxygen and hence reduction of the quantum yields. Perhaps more seriously, photochemical damage by the formation of singlet oxygen through intersystem crossing is reduced by the structure. The tightly constructed can would appear to serve this role nicely says the researchers in Washington, as well as provide overall stability and resistance to unfolding by heat and denaturants.

The location of certain amino acid side chains in a GFP Structure projects the vicinity of the microscopic fluorophore also begins to explain the fluorescence and the behavior of certain mutants of the protein. At least two resonant forms of the microscopic fluorophore can be drawn, one with a partial negative charge on the benzyl oxygen, and one with the charge on the carbonyl oxygen of the imidazolidone ring. However, basic residues appear to form hydrogen bonds with each of these oxygen atoms. These bases presumably act to stabilize and possibly further delocalize the charge on the microscopic fluorophore. Most of the other polar residues in the pocket form an apparent hydrogen-bonding network on the side of a substance that requires abstraction of protons in the oxidation process. It is tempting to speculate that these residues help abstract the protons. A quantitative explanation will require further examination according to the studies and experiments. It seems likely that other mutations of the residues identified to be near the microscopic fluorophore would also have effects on the absorption and/or emission spectra, and such experiments to change the electrostatic environment around the microsopic fluorophore are in progress. By virtue of their varied microscopic fluorophore environments and hence altered spectra, these mutants should lead to expanded uses of green fluorescent protein as gene markers, cell lineage markers, and encourage other uses in biotechnology. Mutations in regions of the sequence adjacent to the microscopic fluorophore, have been systematically explored, some having significant wavelength shifts and most suffer a loss of fluorescence intensity. The mechanism for increased fluorescence may be reduced collisional quenching, as the additional methyl group may make for better packing in the interior of the protein.