Wavelength mutations and posttranslational autoxidation of green fluorescent protein.

TLDR

The green fluorescent protein of *Aequorea victoria* is a unique protein whose chromophore forms via cyclization and oxidation of a Ser‑Tyr‑Gly sequence, and cloning studies suggest this post‑translational modification occurs spontaneously or with only ubiquitous enzymes and reactants. The authors mutagenized GFP and screened for variants with altered spectral properties. The study shows that GFP chromophore formation requires only molecular oxygen, occurring with a ~4‑hour time constant independent of dilution, and that mutagenesis yielding a histidine at position 66 produces a blue‑fluorescent variant, thereby expanding GFP’s utility for dual‑color imaging and FRET applications.

Abstract

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is an unusual protein with strong visible absorbance and fluorescence from a p-hydroxybenzylidene-imidazolidinone chromophore, which is generated by cyclization and oxidation of the protein's own Ser-Tyr-Gly sequence at positions 65-67. Cloning of the cDNA and heterologous expression of fluorescent protein in a wide variety of organisms indicate that this unique posttranslational modification must be either spontaneous or dependent only on ubiquitous enzymes and reactants. We report that formation of the final fluorophore requires molecular oxygen and proceeds with a time constant (approximately 4 hr at 22 degrees C and atmospheric pO2) independent of dilution, implying that the oxidation does not require enzymes or cofactors. GFP was mutagenized and screened for variants with altered spectra. The most striking mutant fluoresced blue and contained histidine in place of Tyr-66. The availability of two visibly distinct colors should significantly extend the usefulness of GFP in molecular and cell biology by enabling in vivo visualization of differential gene expression and protein localization and measurement of protein association by fluorescence resonance energy transfer.