The tightly wound cylindrical fold and the central location of the fluorophore can explain the observed protection of the fluorophore from collisional quenching by oxygen and hence reduction of the quantum yield. Photochemical damage by the formation of singlet oxygen through intersystem crossing is reduced by the structure. Most of the polar residues in the pocket form a hydrogen-bonding network on the side of Tyr66. Atoms in the side chains of Thr203, Glu222, and Ile167 are in van der Waals contact with Tyr66. The mutation of these residues has direct steric effects on the fluorophore.
Mutation also changes the electrostatic environment if the charge is changed.
Furthermore, mutated residues near the fluorophore have direct effects on the absorption and emission spectra. Some mutations have significant wavelength shifts and most suffer a loss of fluorescence intensity. Random and directed point mutations have resulted in various changes in fluorescent behavior. The Ser65 to Thr mutantion increase in fluorescence intensity. The mechanism for increased fluorescence may be reduced collisional quenching.