STRUCTURAL BASIS OF SPECTRAL SHIFTS IN THE YELLOW-EMISSION VARIANTS OF GREEN FLUORESCENT PROTEIN

Background: Because of its ability to spontaneously generate its own f luorophore, the green fluorescent protein (GFP) from the jellyfish Aeq uorea victoria is used extensively as a fluorescent marker in molecula r and cell biology. The yellow fluorescent proteins (YFPs) have the lo ngest wavelength emissions of all GFP variants examined to date. This shift in the spectrum is the result of a T203Y substitution (single-le tter amino acid code), a mutation rationally designed on the basis of the X-ray structure of GFP S65T. Results: We have determined the cryst al structures of YFP T203Y/S65G/V68L/S72A and YFP H148G to 2.5 and 2.6 Angstrom resolution, respectively. Both structures show clear electro n density for nearly coplanar pi-pi stacking between Tyr203 and the ch romophore. The chromophore has been displaced by nearly 1 A in compari son to other available structures. Although the H148G mutation results in the generation of a solvent channel to the chromophore cavity, int ense fluorescence is maintained. The chromophore in the intact protein can be titrated, and the two variants have pK(a) values of 7.0 (YFP) and 8.0 (YFP H148G). Conclusions: The observed red shift of the T203Y YFP variant is proposed to be mainly due to the additional polarizabil ity of the pi-stacked Tyr203. The altered location of the chromophore suggests that the exact positions of nearby residues are not crucial f or the chemistry of chromophore formation. The YFPs significantly exte nd the pH range over which GFPs may be employed as pH indicators in li ve cells.