The GFP chromophore is formed by covalent modification of the three internal residues Ser65, Tyr66, and Gly 67, and consist of a conjugated planar p- system connecting the phenol ring derived from Tyr66 with the imidazolinone heterocycle derived from backbone cyclization of residues 65 and 67 .
YFP sensitivity to these small anions results from ground-state binding near the chromophore, which apparently alters the chromophore ionization constant and hence the fluorescence emission. This effect is observed for all YFPs investigated (YFP, YFP-H148Q, YFP-E222Q, YFP-H148G), although the response decrease in magnitude as the pK a value of the variant increase. The crystallographic investigation presented here demonstrates that in YFP-H148Q, the halide interacts directly with both the chromophore and the p -stacked Tyr203 residue. The absorbance spectrum of YFP is a function of NaCl concentration, with conversion the chromophore anion ( l max 514 nm), to the neutral form ( l max 392 nm) upon addition of chloride. Only the anion is fluorescent in the YFPs, leading to suppression of fluorescence in proportion to loss of band ( l max 514 nm) as [NaCl] is increase . The researchers determined the emission intensity of YFP and YFP-H148Q as a function of NaCl as well as pH (from pH 6.0 to 8.0), under conditions of constant ionic strength. The researcher determined the apparent chloride binding constant (K app ) as a function of pH by fitting the data to a single-site binding model. They determined two crystal structures of YFP-H148Q, one containing two bound iodides, and the other containing no bound halides at all. They found two distinct electron density peaks at 7.7 and 5.5 rms deviations respectively, one located close to the chromophore and buried in the protein interior, the other in a small indentation on the protein surface at the cap of the barrel .