Green Fluorescent Protein - Its Properties And Uses

Nature is the primary source of invaluable biomolecular tools which herald advances in many fields of biology. HindIII, Taq polymerase, and CRISPR-Cas9 are testimony. One of the more seminal tools is GFP, isolated from jellyfish Aequorea victoria. Vital to its importance is the protein`s ability to fluoresce in other organisms without extra cofactors and enzymes, only requiring molecular oxygen (Tsien, 1998). Through using GFP as a reporter molecule, previously obscure biological systems and processes have been visualized.

Wild-type GFP is a 27 kDA protein with an 11-stranded &-barrel conformation (Yang, F. et al., 1996). The chromophore is internal to the barrel and suspended by an &-helix which threads through the centre (Ormo et al., 1996). Because of the tight conformation of the beta barrel, sometimes referred to as a beta can, the chromophore is (1) protected from quenching by water so it does not lose fluorescence, (2) assumed to be formed spontaneously since it is inaccessible to enzymes which would normally catalyse the formation of a fluorophore, and (3) stable since it is mostly resistant to changes in the microenvironment. All three features contributed to wtGFP`s novelty as a reporter molecule. The chromophore was characterized by Cody et al. (1993) as an imidazoline ring formed by cyclization of a post translationally modified tripeptide, 65Ser-66Tyr-67Gly. This tripeptide is sandwiched between three amino acids (64Phe 65Ser 66dehydroTyr 67Gly 68Val 69Gln), the hexapeptide responsible for wtGFP`s fluorescent properties. Maturation of the chromophore itself is contested with two main mechanisms proposed (Figure 2): (A) the cyclization-dehydration-oxidation reaction elucidated by Reid and Flynn (1997) and (B) the cyclization-oxidation-dehydration mechanism described by Barondeau et al. (2003) and (Rosenow et al., 2004). The cyclized peptide is stabilized or trapped through dehydration in mechanism A whereas in mechanism B, by oxidation (Craggs, 2009).

Maximum peak absorbance of wtGFP occurs at 395 nm with a minor peak occurring at 475 nm (Ward and Bokman, 1982). Emission is commonly recorded at 509 nm when triggered by ultraviolet or blue light giving off a characteristic glow seen in A. victoria`s bell margin, later first expressed in a different organism, C. elegans, by Chalfie et al. (1994). Almost simultaneous with the elaboration of wtGFP`s crystalline structure was the start of GFP enhancement by mutagenesis. Target traits for development of A. victoria GFP included emission and excitation peaks, fluorescence brightness, stability, and boosted maturation times. Early modifications were executed by Heim, Prasher, and Tsien (1994-1996) by random mutagenesis and included notable developments: an S65T mutant with longer wavelengths of excitation and emission (489 and 511 nm), blue (BFP), and cyan fluorescent proteins (CFP) produced by Y66H and Y66W mutations respectively. Work on improving the thermostability of GFP at 37-42 C for use in eukaryotic cells included successful attempts by Siemering et al. (1996) and Lim et al. (1995) via point mutations separately made at V163A, S175G, and S147P. Complementing increased thermostability for use in eukaryotic cells was Crameri and company`s (1996) work on improving the intensity of fluorescence of whole cells via DNA shuffling. Notable as well was a study using FACS (Cormack et al., 1996) to characterize three mutants with hundred-fold improvement in fluorescence intensity and increased folding capacity. Perhaps the culmination of research on optimizing wtGFP in 90s was the engineering of EGFP by Yang, T.T. et al. (1998) at CLONTECH Laboratories. Still widely used, EGFP is mutated at F64L and S65T. This engineered protein has an increased folding efficiency at 37 C, one excitation peak at 490 nm, and codons optimized for mammalian hosts.

Following the surge of improvements in the late 90s were several salient modifications to GFP in the 21st century. Patterson and Lippincott-Schwartz (2002) were the first report a photoactivatable T203H GFP variant (PA-GFP) which vastly improved the labelling capacity of GFP on live cells in vivo. In 2006, the original superfold GFP (Pedelacq et al., 2006) was engineered with 5 10-1s-1 rate by S30R and Y39N mutations. So far, mGreenLantern (Campbell et al., 2020) is the brightest and most rapidly maturing variety of GFP by 630% and 207% respectively compared to EGFP.

GFP and fluorescent proteins in general have many applications in both structural and functional studies. Classic applications include use of GFP for biomolecule, cell, and tissue labelling, photobleaching techniques, promoter tracking, subcellular localization, and protein-protein interactions (Zimmer, 2002& Chudakov et al., 2010). Recent remarkable applications of GFP include its use to engineer single cell biological lasers (Gather and Yun, 2014) and synthetic morphogens (Stapornwongkul et al., 2020).

For the past three decades, GFP has served as an imaging workhorse. Continuing work on improving its function will lead to advanced and relevant applications. Additionally, GFP-like proteins extracted from anthozoans is likely to increase the repertoire and flexibility of available tools.