Study on Project Green Light

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The light that comes from it, twinkles like tiny stars in the moonlit water; it shows many reactions at the cell and molecular level; with the aid of it, the microcosmic can be dramatically colorful. It is GFP, Green Fluorescence Protein!

GFP was first discovered and purified from Aequorea victoria by Osamu Shimomura in the 1960s and 1970s respectively. [1] Osamu Shimomura studied the separate luminescent protein Aequorin and found that the GFP's fluorescence occurs by induction of a blue glow which in turn results from interaction between Aequorin and Ca2+ ions. He claimed the reason of GFP's green fluorescence is some kind of luminescent energy that transfers to the GFP. [1]

Fig. 1: Left: Crystal jelly (Aequorea Victoria) Right: A San Diego beach scene drawn with an eight color palette of bacterial colonies expressing fluorescent proteins derived from GFP.

However, at that time, this new interesting protein was underappreciated and nobody thought it would become an important tool in the future. In 1992, Douglas Prasher reported that his group managed to clone the cDNA of wild type GFP and insert it in vector successfully, the using of GFP as a genetic tool for molecular biologists started. [2]

After discovering the sequence of GFP, Frederick Tsuji's lab published a paper about the expression of the recombinant GFP in 1994 independently. [3]And they found that the GFP molecule folds correctly and fluoresces at room temperature without the need of exogenous cofactors.

In 1996, the Remington group was the first group which published the crystal structure of GFP in Science by studying the S65T mutant. [4] In addition, the wtGFP structure was reported in Nature Biotech. by Phillips groups. [5] With studying the structures of GFP, it was clear that the mechanism of its fluorescing is based on the structure of chromophore formation and neighboring residue interactions.

Today, GFP and its derivatives are used universally. The most interesting thing is that the color of GFP can be changed by modifying the chromophore. (Fig.1Right)

GFP has not only brought the light to the microcosm but also brought glorious light to three persons' life. Martin Chalfie, Osamu Shimomura and Roger Y. Tsien shared the 2008 Nobel Prize in Chemistry for their discovery and development of the Green Fluorescent Protein. [6]


GFP is a β-barrel composed of eleven anti parallel β-strands. Two short segments of α -helices form caps at the top and bottom of this structure which resembles a can comprised of 238 amino acids. Approximately near the geometric center of this can shaped structure, there is a chromophore group. (Fig.2). Chromophore is a group of atoms within a molecule which is responsible for the color of molecule. The bonds between the atoms in a chromophore, absorb certain wavelength of light and give off light in different wavelength [7].

Fig.2: Left: Overall structure of GFP. The β-strands are shown in green, α -helices in yellow and chromophore group is in the center [8]. Right: A topology diagram of the folding pattern in GFP [7].

How GFP fluoresce green glow in Aequorea Victoria jellyfish:

Jellyfish has a chemiluminescent protein named Aequorin. When exposed to light, Aequorin involves in a calcium-dependent reaction that leads to the emission of blue light with peak near 470 nm. This wavelength is close to one of the excitation peaks of GFP. Then excited GFP converts blue light to green shine. The molecular mechanism behind this phenomenon is shown in figure 3. Serine 65, tyrosine 66 and glycine 67 are three amino acids of this protein which involve in chromophore formation during three key steps: cyclization, dehydration and oxidation [8], [9].

Fig.3. Left: Mechanism of Chromophore formation in GFP [8]. Right: GFP's chromophore shines green when exposed to the blue light.


GFP provides researchers with a very powerful tool for variety applications that were not feasible a decade ago. One of the important applications of Green Fluorescent Protein is labeling of different proteins to study their localization, dynamics and interactions. The big advantageous of GFP labeling technique in comparison with other labeling methods like using of antibody is that this technique has to be done in living cell. In this technique, a fusion between gene encoding GFP protein and gene encoding our interested protein is made. Therefore, the expressed protein has two subdomains consists of both of these two proteins while GFP does not affect the protein of interest functions but make the protein to be seen in fluorescent microscope. In this way, GFP works as a marker for us to follow the target protein in cell [10]. (Fig.4)

Fig.4. Schematic picture for inserting GFP's gene to target protein's gene.

In this way, GFP can be a useful tool in cancer research. As we know, cancer is a class of cell displays uncontrolled growth, invasion and metastasis to other sites of body. The research group developed an imaging of cancer in animal model in order to visualize cancer cells. Researchers can use labeled cancer cells to study tumor formation, behaviors, and metastases as well as to evaluate the therapeutic effects by different treatment. RM Hoffman group have developed a GFP-expressing cancer cell line which injected into the mouse for tracking metastasis in vivo. by exposed or isolated organs or tissues. Detection of stably expressed GFP in vivo does not require any additional substrate or agent. By using only a 395nm blue light illumination, the GFP-based fluorescent tumor imaging system has become more popular in the cancer research. The researchers injected pancreatic cancer BxPC-3 cell stable expressing GFP into the mice subcutaneously. The image was taken few weeks after injection from whole-body of mouse. It was clearly shown that the tumor and metastases contain GFP report gene can be easily distinguished from other tissues. Tumor growth and formation of metastases could be easily monitored by quantitative analysis of emitted light from GFP [11].

Fig.5: Whole-body images of the BxPC-3-GFP primary tumor (P) and omental (O), bowel (B), and spleen (S) metastases.[11]

Another advantageous of GFP is that we can engineer this protein to make different colors. For example, by changing Tyr of chromophore to His, the color will be changed from green to blue. As a result, instead of labeling just one protein with green label we can lable different proteins in different colors and study their functions simultaneously [11].

Pictures shown below are not masterpieces of modern art, but they are beautiful photographs from genetically modified mouse with fluorescent multicolored neurons created in Harvard Brain Center which is called Brainbow. This strategy for visualizing neural network by using genetically labeled neurons in different colors has potential to revolutionize neurobiology. In this way, researchers would be able to make a precise wiring diagram of the brain which is a promising method for future research on neurodegenerative defects such as Altzheimer's and Parkinson's disease [12]. (Fig.6)

Fig.6. Cerebellar circuit tracing and color analysis[12]

Some researchers have shown that GFP can be used as a biosensor. It is one of the widely used applications of GFP, based on inducing the fluorescence resonance energy transfer. FRET is a kind of energy transference phenomenon between two fluorophores. It can occur when the spectrum emitted by the donor fluorescent molecule overlaps the excitation spectrum of the other fluorescent molecule. [13]

FRET can be dominated via controlling the distance between the two fluorescent molecules that are linked by a short stretch of calmodulin (CaM), a small protein that binds calcium in cells. When the content of Ca2+ is higher than normal level, the additional Ca2+ can combine with calmodulin, to form a Ca2+-CaM complex. This causes a conformational change in calmodulin protein which brings two fluorescent molecules closer together and as a result FRET occurs. The more the level of Ca2+ in the cell, the more FRET occurs, because fluorescent molecules can come closer to each other. This kind of technology can be used as a calcium biosensor which is named Cameleon. [14]


Fig.7: Mechanism of Cameleon as a Calcium biosensor.