Wavelength mutations and posttranslational autoxidation of green fluorescent protein.

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 (-4 hr at 22°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.Proteins are often labeled with fluorescent tags to detect their localization and sometimes their conformational changes both in vitro and in intact cells. Such labeling is essential both for immunofluorescence and for fluorescence analog cytochemistry, in which the biochemistry and trafficking of proteins are monitored after microinjection into living cells (1). Traditionally, fluorescence labeling is done by purifying proteins and then covalently conjugating them to reactive derivatives of organic fluorophores. The stoichiometry and locations of dye attachment are often difficult to control, and careful repurification of the proteins is usually necessary. If the proteins are to be used inside living cells, a final challenging step is to get them across the plasma membrane via micropipet techniques or various methods of reversible permeabilization.An alternative would be to devise molecular biological means to generate fluorescent proteins. The natural UV fluorescence of tryptophan residues is of little use except in the simplest in vitro samples, because the wavelengths are too short and tryptophan is too ubiquitous for any one protein to stand out in a complex biological mixture. A more promising strategy would be to concatenate the gene for the nonfluorescent protein of interest with the gene for a naturally fluorescent protein and express the fusion product. The most highly fluorescent proteins known are the phycobiliproteins (2), but their fluorescence depends entirely on correct enzymatic insertion of a difficult-to-obtain tetrabilin chromophore into a large apoprotein (3). The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a much smaller molecule of 238 amino acids, whose natural function seems to be to convert the blue chemiluminescence of the Ca2+-sensit...

From green to blue: Site‐directed mutagenesis of the green fluorescent protein to teach protein structure–function relationships

“…This deletion produces a protein lacking any fluorescence. The restoration of the Thr at position 64 or the introduction of mutations Y66H or Y66H/Y145F has a dramatic effect on the fluorescent properties of the mutated protein because green fluorescence is restored, or, more importantly, green fluorescence is shifted to blue [12]. Additionally, the mutation Y145F produces a significant increase in the blue fluorescence [1].…”

Development of an in vitro model of excess intracellular reactive oxygen species

“…Considering the nature of GFP folding, this result would be expected. GFP chromophore formation is thought to be dependent on the oxidation of Tyr66 (Heim et al 1994), and requires an increased oxidative state to fluoresce. Reducing agents, such as iron sulfide (FeSO 4 ) and sodium dithionite (Na 2 S 2 O 4 ), convert GFP to a nonfluorescent form, while weaker reducing agents such as b-mercaptoethanol and reduced glutathione, GSH, do not affect its fluorescence (Inouye and Tsuji 1994).…”

Stepwise Simulation of 3,5-Dihydro-5-methylidene-4H-imidazol-4-one (MIO) Biogenesis in Histidine Ammonia-lyase

“…14 In their seminal report on the crystal structure of mature Pp-HAL, Schulz et al 7 postulated a mechanism (Scheme 1, A) that was analogous to that proposed for fluorophore biosynthesis in Aequorea victoria GFP (Av-GFP) by Tsien and co-workers a few years earlier. 15 In both proteins, the imidazolidin-5-one heterocycle would result from the attack of the backbone nitrogen of a glycine (Gly144 in Pp-HAL and Gly67 in Av-GFP) onto the carbonyl carbon of the amino acid two positions before in the primary sequence (Ala142 in Pp-HAL and Ser65 in Av-GFP). Thus, an initial cyclization step (I A ), followed by the elimination of a water molecule, would initially lead to the imidazolidin-5-one ring system (II A ).…”