Structural Biochemistry/Proteins/Fluorescent Marker

Fluorescent markers, sometimes known as fluorophores, is any molecule with the ability to absorb light and emit it at some other well defined wavelength. Flurophores come in a variety of types ranging from simple organic compounds to large proteins like Green Fluorescent Protein. Fluorescent markers give the ability to investigate proteins in their biological environment. When light of a certain wavelength is directed at the molecule's chromophore, a photon is absorbed and excites an electron to a higher energy state. The electron then relaxes back to its ground state. The energy that is released is determined by the formula E = hν, where h is Planck's constant and ν is the frequency of the photon. This energy can then be related back to the wavelength of the emitted photon, which corresponds to a specific color on the visible spectrum. The difference between the absorption and emission wavelength is called the Stokes shift. Large Stokes shifts are generally desirable because the emitted light from the fluorescent tag can be filtered out more easily from the excitation light. Fluorescence educing molecules such as FITC (fluorescein isothiocyanate) are used to stain cells which give the ability to examine these cells under a fluorescence microscope. Multiple fluorescent markers can be used to stain different parts of cell. Fluorescence microscopy can aid in determining the location of specific proteins within a cell. One can even track the movement of these proteins and derive possible functions for these proteins of interest.

Green Fluorescent Protein


Green Fluorescent Protein (GFP) is a fluorescent protein that is found in the jellyfish, Aequoria victoria. In biochemistry, it is frequently used as a marker to monitor gene expression and cell division. The naturally-occurring GFP has a major absorbance peak at 395 nm and a minor absorbance peak at 475 nm. It has an emission peak of 508 nm. This means that when exposed to blue light, it emits photons that give off green light, hence giving it the name Green Fluorescent Protein.

The Green Fluorescent Protein is comprised of 238 amino acid. It has a barrel shape, called a β-can, with 11 β-strands forming the walls of the protein and a α-helix running through the center. The β-can has a diameter of 30Å and a length of 40Å. At the center of the molecule is the hydrophobic fluorophore, which is the part of the protein that is responsible for absorbing and emitting light. The fluorophore is comprised of three amino acids: serine, tyrosine, and glycine, at locations 65, 66, and 67 respectively.

To observe a target protein using fluorescence microscopy, the gene for the production of GFP must be spliced into the RNA transcript that codes for the protein of interest. Wherever the gene is expressed to produce this protein, GFP will be produced along with it. These target proteins will now fluoresce when observed under a fluorescence microscope.

The fluorophore is generated by a sequential mechanism in the catalytic process. No co-factors is required for the activation for the catalysis reaction. The reaction is initiated by a rapid cyclization between Ser65 and Gly67 to form an imidazolin-5-one intermediate, which is followed by a much slower rate-limiting oxygenation of the Tyr66 side chain by O2 on a timescale of hours. Gly67 is required for formation of the fluorophore, no other amino acid can replace Gly in this role. The reaction here is thermosensitive. The yield of formation of the fluorophore decreases when the temperature increase higher than 30oC. Once GFP has produced, GFP turns into thermostable state.

When fluorescent molecules are introduced into a genome, they tend to be phototoxic which can cause death of cells when the fluorescent molecule is active. Majority of small fluorescent molecules tend to have some degree of phototoxicity such FITC (fluorescein isothiocyanate). Because GFP's tri-peptide fluorophore is activated by oxygen, the addition of an enzyme is unnecessary. This oxygen activated characteristic allows the target cell to be less disturbed which causes GFP to be less harmful when used in living cells. This allows GFP to be maintained in the genome.

Another method that can be used to locate a protein is to bind GFP to an antibody that will bind to the target protein. The direct method would bind the fluorescent antibody to the corresponding antigen on the target protein. The indirect method first binds a non-fluorescent antibody on the target protein. Then the fluorescent antibody is introduced which will bind to the non-fluorescent antibody. Although it is possible to stain specific parts of cell directly, indirect methods are preferred due to high affinity. Then using fluorescence microscopy, the target cell which is now fluorescent can be studied.

Other Uses of GFPs
The use of GFP is not only limited to monitoring gene expression and cell regulation. Inorganic species such as zinc and nitric oxide are important agents that help drive physiological processes. Therefore, it is valuable to study and visualize how these inorganic agents interact and are processed within cells. For zinc, there are many turn-on sensors that offer good methods to visualize zinc ions inside cells. Most zinc indicators used are intensity-based sensors such that the response to zinc indicators establishes the intensity of fluorescence emission. Nitric oxide, on the other hand, are more difficult to monitor because of their gaseous nature, limited water solubility, and other limitations. One way to work around this challenge is to use genetic encoding of nitric oxide-sensitive proteins which have transition metal nitric-oxide reactive sites. For example, two mutant GFPs are fused together to be used as a nitric oxide indicator. In a sense, one can manipulate GFPs to use as indicators, thereby giving even more possibilities to understand organization and regulation of signaling networks.

Green fluorescent proteins are also used in the in vivo technique of FRET (Fluorescent Resonance Energy Transfer), an imagining method used to understand interactions between proteins. In FRET, the energy transfer from a fluorescent protein (the "donor") to another fluorescent protein with a longer wavelength (the "acceptor") are measured. By measuring the inter-molecular and intra-molecular distances between the GFP bounded proteins, researchers are able to observe and take note of protein interactions. For example, FRET allows us to detect interactions between signaling molecules. By situating a protein that is sensitive to changes in conformations between the FRET donor and acceptor proteins, we can determine the activity of the pathway.

Fluorescent Sensors for Nitric Oxide
Metal-based fluorescent probes have been developed as method for detecting the presence of NO in a system; NO is a prominent compound that is associated with signaling pathways in the living organisms. These metal-based fluorescent indicators are based on the metal Cu(II). When the Cu(II) ions react with NO, Cu(II) gets reduced into Cu(I). This process results in diminishing the quenching energy of the lone pair on the nitrogen atom of the nitric oxide; the reaction causes a decrease in the fluorescence intensity of the lone pair on the nitrogen atom. In addition, the react between Cu(II) and NO that takes place in the system also leads to a lowering of the PET quenching in the N-nitrosamine. Fluorescent probes based on Cu(II) are ideal for detecting NO that reside in environments that do not contain oxygen. A specific fluorescein dye that utilizes Cu(II) ions for identifying NO is the CuFL1 platform. The fluorescein dye in this particular scaffold has an aminoquinoline,a derivative of quinoline, which binds to Cu(II) and forms a 16-fold emission turn on. This reaction converts the secondary amine of the aminoquinoline into an N-nitrosated product, which reinforces the fluorescence of the molecule; the increase in the intensity of fluorescence is based on the concentration of NO. The presence of NO in the Raw 264.7 macrophages and certain gram positive cells such as Bacillus subtilis and Bacillus anthracis has been identified with the CuFL probe.

Fluorescent Markers in DNA Sequencing
Fluorescent markers are often used in DNA studies to determine the sequences of bases in a strand. The upside to this method is that it does not utilize radioactive reagents as in the auto-radiography seen in Western blotting. As a result it has become a very popular method of DNA sequencing. In a method devised by Frederick Sanger and colleagues, DNA polymerase was used to create complementary strands to a single strand DNA molecule in four mixtures of radioactively labeled nucleotides. Incorporated into this synthesis was a mixture containing the 2',3'-dideoxy analog of a nucleotide, a different analog for each mixture. The 2',3'-dideoxy analog in the mixture causes the termination of DNA replication creating fragments. This is due to the analog having no 3' hydroxyl terminus to promote phosphodiester bonding. The mixtures then underwent electrophoresis allowing base sequences to be read through autoradiogram. With the use of fluorescent markers, a different colored tag is attached to the dideoxy analog of each base instead of having to be radioactively labeled. The fragments then undergo the same electrophoresis. The resulting bands are detected by their fluorescence and the sequence of colors subsequently translates into the sequence of the bases. With this method more than 1 million bases can be sequenced per day with modern sequencing tools.

Fluorescent Protein Reporters
Strategies are being developed for fluorescent protein (FP) reporters that "speak" the language of the cell and allows scientists to understand complex networks of biochemical processes that they were unable to witness before. There are three main types of FP's that are made currently. The first are ones that are bound to a protein to report its location and turnover, the second are ones created to undergo changes in fluorescent signal to allow scientists to know when there are changes or intermolecular interactions with the protein they are studying. Lastly, there are FP's that contain a sensory element which detects the accumulation or degradation of small molecules. These FP reporters are then used to understand the behavior of tagged signaling molecules and their spatiotemporal organization.

In a normal signaling pathway receptors on the plasma membrane detect extracellular cues and mediate production of intracellular second messengers, these second messengers then regulate the activity of signaling enzymes and downstream transcription factors. These reporters allow scientists to see how a protein reacts in its natural environment, whether it diffuses across a membrane or needs a receptor on the membrane to send a message. These can be observed using a GFP whose fluorescence changes color with time or are photoactivatable, This method allows for direct showing of molecules within cells responsible for the action being observed. The best part of FP's is their ability to remain discrete and not disturb pathways of cell function.

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