Protein Project: Green Fluorescent Protein

The 2008 Nobel Prize in Chemistry was awarded to Osamu Shimomura, Martin Chalfie

and Roger Y. Tsien for the discovery and development of the green fluorescent protein (GFP).

Composed of 238 amino acids, the protein is known for its bright green fluorescence upon

exposure to blue or ultraviolet light. Shimomura first extracted the protein from the jellyfish

Aequorea victoria but similar fluorescent proteins have been exhibited in other marine organisms

(Tsien, 1998). In Aequorea victoria, GFP serves as an accessory protein that converts the blue

light emitted by the aeqrorin, a photoprotein, to green light (Sullivan, 1999). The protein’s

amazing ability to generate highly visible internal fluorophores on its own leads to its common

use in molecular and cellular biology research as a reporter of gene expression and as a

biosensor. Recent research with the protein has involved developing alternatives with different

colors and enhanced fluorescence.

GFP is one of the first proteins whose structure has been extensively studied in relation

to its spectroscopic function. The structure of the 238-residue protein consists of an 11-stranded

anti-parallel -barrel with the chromophore located at the geometric center of the -barrel on an

-helix in the threaded through the barrel. Additionally, the protein includes small sections of a-

helix that form caps at both ends of the -barrel cylinder. This structure, shown in Figures 1 and

2, is termed the -can (Phillips, 1997). The synthesis of the GFP fluorophore occurs through an

autocatalytic process without the use of cofactors or accessory proteins. Amino acids central to

the intramolecular cyclization mechanism of fluorophores generation include serine, tyrosine,

and glycine located at positions 65-67 within the peptide. The presence of molecular oxygen is

required for the proper folding and subsequent chromophore formation. The first step in the

formation of the fluorophore following protein folding is the formation of an imidazolinone by a

nucleophile attack by the amide on the glycine residue on the carbonyl group of the serine reside.

The reaction is followed by the dehydration of the carbonyl oxygen of the serine residue. The

final and rate-limiting step in the development of the fluorescence is the oxidation of the

hyroxybenzyl side chain of Tyr-66 by oxygen to put its aromatic group in conjugation with the

imidazolinone ring and to produce the final fluorescent product p-

hydroxybenzylideneimidazolinone (Barondeau, 2003). This process is depicted in Figure 3.

The proper folding and structural characteristics of GFP are crucial to its observable

bioluminescent properties. The wild-type GFP has a major excitation peak at 395 nm and a

minor peak at 475 nm (Phillips, 1997). The two excitation wavelengths produce emission peaks

at 508 nm and 503 nm respectively (Tsien, 1998). The fluorophore formation process is highly

thermosensitive and yield of fluorescent protein decreases at temperatures greater than 30 °C.

Once GFP has been post-translationally modified, however, the molecule is relatively

thermostable. The tightly packed -barrel structure provides the fluorophores inside stability and

resistance to denaturation. The -can structure protects the chromophore from quenching by

molecular oxygen or attack from hydronium ions (Zimmer, 2002).

Since its discovery, GFP has become a common detection and imaging tool in molecular

and cellular biology. GFP’s wide use is largely due to its unique fluorescence properties,

autocatalytic fluorphore formation, minimal effect on activity of conjugated proteins and

biocompatibility. One frequent application of GFP allows researchers to track protein movement

within a cell by fusing GFP with a protein of interest. Using imaging techniques, the location of

GFP and the attached protein can be monitored over time. In most reported studies, GFP does not

have an effect on the function of proteins being tracked and therefore can be successfully applied

to these protein studies. GFP can also serve as a reporter protein for the determination of gene

expression. If the gene for GFP is inserted into a genome after a promoter, the fluorescence of

GFP can be used to determine the transcriptional activity of the promoter and the genes

following it. The use of GFP allows for gene expression to be assayed in a quantitative manner.

While many other assays require the cell to be lysed, the use of a fluorescent tracker allows for

the determination of expression over time and in a noninvasive manner (Misteli, 1997). GFP has

also allowed researchers to understand how cancer metastasis and growth occurs. Yang, et. al

inserted cancerous cells with GFP into mice and were able to image the malignant growth and

spread in live mice. This technique has been used in many subsequent studies to test potential

chemotherapeutic agents (Yang, 2000). As the emission of GFP is sensitive to changes in pH,

calcium ion concentration and temperature, many researchers have utilized these properties to

develop GFP variants as molecular sensors (Donner, 2012; Bizzarri, 2009; Tsein, 1998).

One recent development in the use of GFP as a biosensor has been to allow more accurate

identification of protein levels in human serum. As protein imbalances in serum are a good

indication of disease states, the detection of biologically relevant changes could improve

diagnostics. De, Rana, Akpinar, et. al. created a nanoparticle (NP)-GFP hybrid sensor with an

identification accuracy of 97% in serum. Distinct fluorescence patterns are generated in

response to proteins such as serum albumin, immunoglobulin G, transferrin, fibrinogen, and α-

antirypsin using five different complexes. The GFP-NP conjugates have the ability to mimic

protein-protein interactions, allowing this system to detect protein with lower concentrations than

previous methods (De, 2009).

Given the wide range of potential applications for GFP, many efforts have been made to

optimize the thermal and fluorescent properties through the engineering of mutants. Roger Heim

and Roger Tsien proposed some alterations in the wild-type GFP that resulted in improved

fluorescence and allowed for new spectral properties. Development of mutant GFPs with

different excitation and emission spectra allows for the detection of multiple cellular events at

the same time. They observed that replacing the tyrosine in position 66 with a histidine or a

tryptophan residue resulted in decrease in wavelength of both the excitation and emission peaks.

They were able to optimize the fluorescence of these blue fluorescent proteins by further

mutating the protein at residues 145-163, believed to be close to the fluorophore upon protein

folding. Additional mutational combinations resulted in three distinguishable varieties of GFP

(Heim, 1995). Colored mutations of GFP have lead to the exploration of their potential to

participate in fluorescence resonance energy transfer (FRET) in recent years. FRET is the

nonradiative exchange of energy from an excited donor fluorophores to a nearby acceptor

fluorophore. Interactions between two proteins linked with GFP mutants with differing spectral

ranges can be studied by measuring the FRET interactions between the pair of mutants. This

technique is used for the calcium biosensor application of GFP as the protein calmodulin brings

two GFP mutants close together upon calcium binding and increases FRET interaction between

the pair (Zimmer, 2002). The thermoselectivity of GFP also limits the possible applications that

require higher temperatures but mutants have been developed to extend the functional

temperature range of GFP (Sullivan, 1999).

The discovery of GFP by Osamu Shimomura and its subsequent development by Martin

Chalfie and Roger Tsien has introduced a ubiquitous and versatile technology to molecular and

cellular biology. It has allowed a method for scientists to track proteins and various molecules in

a noninvasive method over time. GFP has generated much interest outside of the scientific

community as artists genetically incorporate GFP into animals to create visually striking images.

The use of GFP in biochemical studies is likely to expand in the coming years as we are able to

further modify the properties of GFP to suit these applications.

Figure 1. 3-Dimensional representation of Wild-Type Green Fluorescent Protein

Figure 2. Structure of Green Fluorescent Protein

Figure 3. Process of Fluorophore Formation

Works Cited

1. Tsien, R. (1998). The Green Fluorescent Protein. Annual Review of Biochemistry, 67,

509-544.

2. Barondeau, D., Putnam, C., Kassmann, C., Tainer, J., & Getzoff, E. (2003). Mechanism

and energetics of green fluorescent protein chromophore synthesis revealed by trapped

intermediate structures. Proceedings of the National Academy of Sciences, 100(21),

12111-12116.

3. Sullivan, K., & Kay, S. (1999). Green fluorescent proteins. San Diego, California:

Academic Press.

4. Phillips, G. (1997). Structure and dynamics of green fluorescent protein. Current Opinion

in Structural Biology, 7, 821-827.

5. Zimmer, M. (2002). Green Fluorescent Protein (GFP):  Applications, Structure, And

Related Photophysical Behavior. Chemical Reviews, 102, 759-781.

6. Heim, R., & Tsien, R. (1995). Engineering Green Fluorescent Protein For Improved

Brightness, Longer Wavelengths And Fluorescence Resonance Energy Transfer. Current

Biology, 67, 178-182.

7. Yang, M., Baranov, E., Jiang, P., Sun, F.-X., Li, X.-M., Li, L., … Hoffman, R. M.

(2000). Whole-body optical imaging of green fluorescent protein-expressing tumors and

metastases. Proceedings of the National Academy of Sciences of the United States of

America, 97(3), 1206–1211.

8. Misteli, T., Spector, D. (1997). Applications of the green fluorescent protein in cell

biology and biotechnology. Nature Biotechnology, 15, 961-964.

9. Bizzarri, R., Serresi, M., Luin, F., Beltram, F. (2009) Green fluorescent protein based pH

indicators for invivo use: a review. Analytical and Bioanalytical Chemistry, 393, 1107-

1122.

10. Donner, J. S., Thompson, S. A., Kreuzer, M. P., Baffou, G., Quidant, R. (2012) Mapping

Intracellular Temperature Using Green Fluorescent Protein. Nano Letters, 12(4), 2107-

2111.

11. De, M., Rana, S., Akpinar, H., Miranda, O. R., Arvizo, R. R., Bunz, U. H., Rotello, V. M.

(2009) Sensing of proteins in human serum using conjugates of nanoparticles and green

fluorescent protein. Nature Chemistry. 1, 461-465.