Chemical nature of the light emitter of the Aequorea green fluorescent protein

Abstract

The jellyfish Aequorea victoria possesses in the margin of its umbrella a green f luorescent protein (GFP, 27 kDa) that serves as the ultimate light emitter in the bioluminescence reaction of the animal. The protein is made up of 238 amino acid residues in a single polypeptide chain and produces a greenish f luorescence ( max ‫؍‬ 508 nm) when irradiated with long ultraviolet light. The f luorescence is due to the presence of a chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser 65 -Tyr 66 -Gly 67 -. GFP has been used extensively as a reporter protein for monitoring gene expression in eukaryotic and prokaryotic cells, but relatively little is known about the chemical mechanism by which f luorescence is produced. To obtain a better understanding of this problem, we studied a peptide fragment of GFP bearing the chromophore and a synthetic model compound of the chromophore. The results indicate that the GFP chromophore consists of an imidazolone ring structure and that the light emitter is the singlet excited state of the phenolate anion of the chromophore. Further, the light emission is highly dependent on the microenvironment around the chromophore and that inhibition of isomerization of the exo-methylene double bond of the chromophore accounts for its efficient light emission.

Thermal Effect on Aequorea Green Fluorescent Protein Anionic and Neutral Chromophore Forms Fluorescence

“…The bands visible at 400 nm and 476 nm are usually associated with the chromophore protonation states neutral (N) and anionic (A), respectively [15,23]. The excitation band at 278 nm is due to nonspecific, more energetic electronic transitions occurring in the chromophore.…”

“…Regarding its photophysics, the wild type GFP protein is characterized by high quantum yields, ϕ f~0 .8, in contrast to what is observed for its individual cromophore in aqueous solution, ϕ f~1 0 −3 [14,15]. In the native fold, the inhibition of molecular vibration or rotation along the chromophore double exo-metilene bond, prevents emission quenching by a rapid internal conversion process [13,15,16].…”

“…In the native fold, the inhibition of molecular vibration or rotation along the chromophore double exo-metilene bond, prevents emission quenching by a rapid internal conversion process [13,15,16]. Inside the β-barrel, chromophore's fluorescence extinction by O 2 [17] and by hydron ions [18] is also avoid.…”

Label-free Raman observation of cytochrome c dynamics during apoptosis

“…The label-free observation has advantages especially in observation of dynamics of biomolecules because labeling markers may disturb the motion of target molecules with a size and weight greater than or comparable to the target. For example, cytochrome c consists of 104 amino acids and weighs about 12.4 kDa, whereas a typical GFP consists of 238 amino acids and weighs about 27 kDa (19,20). Raman imaging can also contribute to the observation of molecule-specific tags with reduced labeling effects by using Raman-active molecular tags smaller than the target, such as deuterium or alkynes (21,22).…”

Quenching of superoxide radicals by green fluorescent protein

“…Additionally, another potent antioxidant found in many cnidarians is the imidazole compound coelenterazine, a Ca 2+ dependent luciferin used as a substrate for bioluminescence in many marine species [45]. There is remarkable similarity in the structure of coeleterazine and GFP [46], and like GFP in corals coeleterazine is found in several cnidarians without bioluminescense [45]. Both theoretical and experimental data suggest that the luciferin/ luciferase enzyme system first evolved as an ROS scavenging system and then was co-opted (i.e., exapted) for light production [45].…”