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 microcosmos can be dramatically colorful. It is GFP, Green Fluorescent Protein!
GFP was first discovered and purified from Aequorea victoria by Osamu Shimomura in the 1960s, and then described during 1970s.[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 is the result of the interaction between aequorin and Ca2+ ions. He claimed that the reason to GFP's green fluorescence is that some kind of luminescent energy is transferred to the GFP. [1]
Fig.1: Left: Crystal jelly (Aequorea Victoria) Right: A colorful culture produced by bacterial colonies expressing different engineered fluorescent proteins.
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 successfully insert it into a vector. Thereby, the use GFP as a genetic tool for molecular biologists started. [2] After discovering the sequence of GFP, Frederick Tsuji's lab, in 1994, independently published a paper about the expression of recombinant GFP independently. [3] 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 that published the crystal structure of GFP in Science, after studying the S65T mutant. [4] In addition, the wtGFP structure was reported in Nature Biotechnology by Phillips. [5] By studying the structure of GFP, it become clear that the mechanism of its fluorescence is based on the structure of the chromophore , 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 microcosmos but also brought glorious light to three persons' life. In the 2008, committee award Martin Chalfie, Osamu Shimomura and Roger Y. Tsien Nobel Prize in Chemistry to praise the success in their discovery and improvement of the Green Fluorescent Protein. [6]
Structure
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. Near the geometric center of this can-shaped structure, lies a group of atoms forming a chromophore (Fig.2). The bonds between the atoms in the chromophore absorb certain wavelengths of light and give off light in a different wavelength [7]. The chromophore is thereby responsible for the molecules color.
Fig.2: Left: Overall structure of GFP. The β-strands are shown in green, α-helices in yellow and the chromophore is at the center [8]. Right: A schematic drawing of GFP's folding pattern [7].
How GFP fluoresce green glow in Aequorea Victoria
Aequorea Victoria has a chemiluminescent protein named aequorin. When exposed to light, aequorin is involved in a calcium-dependent reaction that leads to the emission of blue light with a wavelength peak near 470 nm. This wavelength is close to one of the excitation peaks of GFP, and therefore, the 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 the three amino acids that are involved 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.
Applications
GFP provides researchers with a very powerful tool for a variety of 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 advantage of the GFP labeling technique in comparison with other labeling methods, like the use of antibodies, is that this technique can be used on living cells. In this technique, a fusion, between the gene encoding GFP and gene encoding the target protein, is made. Therefore, the expressed protein has two sub-domains consisting of both of these two proteins. While GFP does not affect the target protein's functions, it enables it to be seen with a fluorescent microscope. In this way, GFP works as a marker for us to follow the target protein in the cell [10] (Fig.4).
Fig.4: Schematic picture for inserting GFP's gene into the target protein's gene.
In this way, GFP can be a useful tool in cancer research. As we know, cancer can be described as a group of cells that displays uncontrolled growth, invasion and metastasis to other sites of the body. Some research groups developed a method for imaging cancer in an animal model, in order to visualize the cancer cells. The labeled cancer cells can be used for studying tumor formation, behaviors, and metastases as well as to evaluate the therapeutic effects by different treatment approaches. RM Hoffman's group developed a GFP-expressing cancer cell line and injected it into exposed or isolated organs or tissues of mice, for tracking metastasis in vivo. Detection of stably expressed GFP in vivo does not require any additional substrate or agent. Since the only thing needed is a 395 nm blue light illumination, the GFP-based fluorescent tumor imaging system has become more popular in cancer research. The researchers injected pancreatic cancer BxPC-3 cells, expressing GFP, into the mouse subcutaneously. The image shown below, displaying the whole body of the mouse, was taken a few weeks after injection. It is clearly shown that the tumor and metastases, that contain the GFP reporter gene, can easily be distinguished from other tissues. Therefore, tumor growth and formation of metastases can easily be monitored by quantitative analysis of emitted light from GFP [11].
Fig.5: Whole body picture of mouse that is injected by BxPC-3-GFP cell shows cancer metastases under blue light, primary tumor (P) and omental (O), bowel (B), and spleen (S) metastases.[11]
Another advantage of GFP is that we can engineer this protein to make different colors. For example, by changing tyrosine 66 of the 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 label different proteins in different colors and study their functions simultaneously [11].
Pictures shown below are not masterpieces of modern art; they are beautiful photographs from genetically modified mice, with fluorescent multicolored neurons, created at Harvard Brain Center. This strategy for visualizing neural networks by using genetically labeled neurons in different colors, referred to as Brainbow, 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 Alzheimer's and Parkinson's disease [12] (Fig.6).
Fig.6.Color analysis of cerebellar circuit [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). FRET is a kind of energy transference phenomenon that occurs 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, 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 is used for a calcium biosensor, named Cameleon. [14]
calmodulin
Fig.7: Mechanism of Cameleon as a Calcium biosensor.
