Structural Biochemistry/Fluorescent Proteins

Definition of Fluorescence

Fluorescence is the process where light, such as visible light, is absorbed by a molecule and re-emitted at a longer wavelength to generate distinct colors. It is a physical phenomena based on excitation of electrons in an atom or molecule; and emission is at a longer wavelength. Fluorescence allows us to see how things interact in a cell, as well as the localization and pathways taken.

Introduction

Fluorescent proteins were first discovered through extraction from the jellyfish Aequorea Victoria. They have been instrumental to studies of cellular biology. Fluorescent proteins contain various color variants which emit various colors at different wavelengths thus functioning as valuable probes that can be used for live cell imaging. The proteins can be used as markers in vivo for whole-body imaging and detection of cancer as well as in organelles where protein fusion could be done to monitor intracellular dynamics and aspects of transcription. The important aspect that contributes to the fluorescent properties of the protein is its structure which consists of various amino acids depending on the protein and the local microenvironments. A variety of derivatives of fluorescent proteins have been created for the use of various markers with the most widely used being Green Fluorescent Protein, which was the topic for which Roger Tsien, a scientist from UCSD, received the Nobel Peace Prize.

Structure

Understanding the protein structure allows one to understand further protein function. This is the case for the structure of fluorescent proteins. For green fluorescent proteins, it is made up of a beta barrel structure, which consists of a β-Helix and alpha helices and which surrounds the fluorophore. The fluorophore is a part of the molecule responsible for its color. The fluorophore for the helix is formed by three amino acids that form a tripeptide Ser65–Tyr66–Gly67. The cyclicization of the amino acid residues is what forms the imidazolidone ring. However, an important property of the tripeptide is that the amino acid sequence is not the intrinsic property that leads to fluorescence as the same amino acid sequence is found in other proteins that do no have fluorescence. Further oxidation of imidazolidone ring causes the conjugation of the ring with Tyr-66 contributing the maturation of protein in terms of fluorescence. A key component of the fluorophore also is the fact that it is in two states. One state of the fluorophore is the protonated state or the predominated state, which has an excitation maximum of 395 nanometers. The other state of the fluorophore is the unprotonated state, which has an excitation maximum of 475 nanometers. Because of the complexity of the fluorophore for green fluorescent proteins, the molecule can accommodate modification. One feature that is significant is the packing of amino acid residues in the beta barrel is stable resulting in a high fluorescent quantum yield. The tight protein structure contributed by H-bonding also has resistance to pH, temperature, and denaturants such as urea.

Variations in Fluorescent Proteins

Yellow Fluorescent Proteins

These fluorescent proteins are represented by the mutation in one of the amino acid residues of the fluorophore. The tyrosine found in Green fluorescent proteins (GFP) is found to have an mutation, which results in the stabilizing of the dipole movement and the shift in wavelength of the excitation/emission spectra resulting in the yellow fluorescence. Furthermore, the yellow fluorescent proteins was found to have a new threonine residue 203 near the fluorophore. Further modification of the yellow fluorescent protein, can enhance the brightness of the protein, which makes it a Enhanced Yellow Fluorescent Protein (EYFP). Because of the brightness of the fluorescence, the protein is an important tool in multicolor imaging. These proteins are derived from the jellyfish Aequorea Victoria and is modified to create a different emission of fluorescence different from the original Green Fluorescent Protein.

Yellow fluorescence protein TagYFP is a bright yellow fluoresence recommended for protein labeling in protein localization and interaction studies. It may also be used for cell and organelle labeling and for tracking promoter activity, although TurboYFP and Phl-Yellow proteins are preferred. It is a monomeric protein successfully used for fusions and was developed under the bassis of GFP-like protein from the jellyfish Aequorea macrodactyla. TagYFP has a fast maturation phase along with high pH-stability and photstability. It's high pH stability allows it to be more stable that EYFP while its fast maturity allows it to give a brighter fluorescent signal. TagYFP has also been proven to generate stable transfected cell lines.

Blue and Cyan Fluorescent Proteins

Blue and Cyan Fluorescent Proteins result from the modification of the Tyr 66 residue located in the fluorophore. The conversion of the Tyr to histidine leads to the emission of blue fluorescence at a wavelength of 450 nanometers. Another modification is the conversion of Tyr to tryptamine resulting in a different fluorescence at a wavelength of about 480-500 nanometers. Additionally, genetic markers which uses these fluorescent proteins are expressed as TagBFP. One of the advantage of this protein is the ease at which it can be express and detect in a wide range of organisms. Mammalian cells transiently transfected with TagBFP expression vectors give bright fluorescent signals within 10-12 hrs after transfection. No cell toxic effects and visible protein aggregation are observed. TagBFP performance in fusions has been demonstrated in the β -actin and α-tubulin models. It can be used in multicolor labeling applications with green, yellow, red, and far-red fluorescent dyes. A problem though of these modifications is that that they require secondary mutations to increase not only folding efficiency, but also brightness. Fortunately, the genome modification is not life-threatening.

Red Fluorescent Proteins

Various derivatives of Red Fluorescent proteins have been derived in hopes to find a protein that exceeds or equals the fluorescence ability of GFP (green fluorescent protein). One derivative of protein has been from the coral Dicosoma striata also known DSRED. When it is matured, the protein is found to have an emission spectrum of about 583 nanometers. Further modification of DSRED has led to the formation of DSRED2 which have mutations at the peptide terminus preventing formation of protein aggregates and reduce toxic levels. Also, the DSRED2 is found to be more compatible with GFP as well. These proteins are also represented by its tetramer structure.

Astrocytes stained for GFAP, with end-feet ensheathing blood vessels

'Green Fluorescent Proteins' The green fluorescent protein (GFP) is derived from about 200+ amino acid residues. This protein will exhibit green fluorescence when under to blue light. This GFP is mainly isolated from a marine biological organism: the jellyfish Aequorea victoria. This GFP has three main excitation maximums. An intense one at wavelength 395, a smaller one at 475 nm, and the emission peak which is at 509 nm which is what gives this protein a distinct vibrant green color. Another organism from the sea pansy has another excitation peak around 498 nm. This GFP gene is important for biosensing and reporting locations of gene expression. It can be transformed into other organism's genome through breeding, invitro injection, or through transformation. The GFP gene is introduced into a variety of bacteria, yeast, fungi, and other multicellular organisms. The story of how GFP became a research tool began in 1992, when Martin Chalfie of Columbia University showed that the gene that makes GFP produced a fluorescent protein when it was removed from the jellyfish genome and transferred to the cells of other organisms. Chalfie, a developmental biologist, first put the gene into bacteria and roundworms, creating glowing versions of these animals. Martin Chalfie, Osamu Shimomura, and Roger Y. Tsien were awarded the 2008 Nobel Prize in chemistry on 10 October 2008 for their discovery and development of the green fluorescent protein. Since then, researchers have transferred the GFP gene into many other organisms and even human cells growing in a lab dish.The GFP is unique amongst natural pigments for its ability to autocatalyse its own chromophore, through atmospheric conditions. In this way, a single protein acts as both substrate and enzyme. Other natural pigments require multiple enzymes for their production. Biotechnology has taken advantage of this unique feature of GFP, putting it to use as an in vivo marker of gene expression and protein localisation. Monomeric Fluorescent Protein Variants

Fluorescent Proteins originally in its natural states exist as dimers, tetramers, and oligomers. Also, the theoretical possibility of fluorescent proteins forming dimers in cellular compartments due to possible high protein concentrations contributing to dimerization. Thus, the use of monomeric fluorescent protein variants has been sought after. One problem however is that the first few monomeric fluorescent proteins had reduced fluorescence capability. Furthermore, the production of the monomers required around 30 amino acid changes to the structure. There are improvements though in the development of these proteins that have increased quantum yields and photostability.

Function

Fluorescent tags are used to label molecules such as proteins, DNA, and antibodies. Fluorescent labeling works with fluorophore reacting with a functional group on the target molecule. Probes are produced from this type of molecular labeling. Western blot assays identify and separate proteins therefore these fluorescent tags are crucial. Size exclusion chromatography removes fluorophore on the target molecules. Fluorescent dyes can be used to specify which organelle of the cell is present in order to distinguish their unique structure and further explore their individual functions. These tags and dyes are important in microscopy and reverse photobleaching because they are less harmful to living cells than quantum dots. Fluorescent dyes have hydrophobic properties hence specific dye columns are used in separations of molecules with dyes.

Example: Western Blot Assay

The purpose of a Western Blot is to locate and determine proteins on the basis of their ability to bind to certain antibodies. A Western Blot analysis allows one to detect a protein of interest from a mixture of a number of proteins. After completing a Western Blot, the information gained from the process is the size of the protein and the expressed amount of protein. A Western Blot analysis can be done on any protein sample, ranging from cells to tissue, to recombinant proteins synthesized in vitro. A condition the Western Blot is dependent on is the quality of the antibody that is used to probe for the target protein. The antibodies used in a Western Blot must be specific to the protein of interest.

Example: Green Fluorescent Protein (GFP) turns green when exposed to blue light. on GFP

Uses

X-Ray Fluorescence Microscopy:

Hard X-Ray fluorescence microscopy is useful to investigate the trace metal distributions within a whole, unstained, biological tissues.

Trace metal elements are integral to many life forms. Metals help catalyze functions and sometimes even play a structural role within the cell. Take for example zinc finger domains, where Zinc ions help to bind nucleic acids and proteins. Metals are recognized for having influential effects on human health and disease. Therefore, the study of these trace elements can provide important information and reasoning for the functions and pathways of metalloproteins. These studies may even find therapeutic approaches to quantitatively study the intracellular distribution of these trace elements.

These X-ray fluorescence has been used to create tomographs to visualize the structure of a 10-μm cell. Despite the usefulness of using these high penetration X-rays for tomography, there have been limitations that affect the length of time of experiments as well as the accuracy of these images. Due to these limitations in X-Ray fluorescence microscopy, there have been ongoing research to develop better X-ray resolution, detector speed, cryogenic environments, and the pursuance of a auxiliary signals. Thus, there will be many helpful new approaches in X-ray fluorescence tomography in the future.

Hard X-ray fluorescence, also called XRF, microscopy is a useful tool to trace metal distributions in various biological systems. For transition metals such as copper, zinc, and other relevant trace elements, XRF has provided attogram sensitivity at spatial resolutions down to 150 nm. Nowadays, 10-15 elements have been able to be mapped simultaneously. This simultaneous mapping of elements leads to precise elemental colocalisation maps.

Structural visualization has been improved even further with the use of hard X-ray fluorescence, but several technical challenges have presented themselves to lead to two-dimensional and low-definition realizations. Current developments have allowed scientists to overcome some of the most major limitations, and now the scientific world is able to create sub-500-nm resolution XRF tomography. Tomographs with this resolution can provide extreme detail in the realm of elemental specificity. These recent progresses in XRF tomography will definitely be utilized soon. Other developments in progress are cryo-microprobges, which are able to accommodate frozen-hydrated specimens.

The process of X-ray fluorescence is extremely fit for quantifying trace elements. XRF is unique because it does not rely on artificial dyes or flurophores to determine the structure of proteins, rather, X-ray fluorescence can be excited by exposing molecules to particles (electrons and protons) or X-ray beams. With the use of X-ray excitation, the bremsstrahlung background becomes unnecessary, allowing WRF microscopy to show high spatial resolution. Because X-ray penetration enables scientists to have sample thicknesses of tens of microns, XRF microscopy is an ideal method for form tomographic visualization of biological samples.

Due to many technical challenges and the analytical complexities involved in XRF tomography, the method has not found general application. Regardless, recent developments have provided advancements in 3D resolutions down to a few 100 nm for specimen up to 10 µm in size. This means 3D elemental maps of multiple elements can be visualized with high spatial resolution. There is a bright future ahead for XRF with its recent advancements. Combined with the demand from the biological, environmental, materials, and geological communities, XRF seems very promising.

XRF Tomography:

The term ‘tomography’ is derived from Greek and means ‘slice imaging’, meaning that it is a technique that gets its data from a single slice within its specimen. A 3D reconstruction is made of slices of a specimen from tilted angles. The task of reconstruction can be quite strenuous and time consuming, given that serial sectioning is the direct approach to tomography. This direct method of actually physically sectioning can lead to significant artefacts, which is a defined as a man-made object that taken as a whole. As an alternative to physically sectioning, non-destructive techniques can be utilized because they reduce specimen preparation requirements and are also significantly easier and simpler to perform. In comparison to other methods of tomography, only XRF microtomography is able to map trace elemental distributions in direct relevance to biology.

Full-field tomography:

Recently, X-ray fluorescence tomography has been demonstrating by using full-field imaging and structured detector approaches. However, these novel full-field imaging and structured detector approaches have their pros and cons as well. These nascent technologies have spatial resolution between 2 and 200 μm, an sensitivity levels between 100ppm to percent levels. They also have a wide range of different elemental contrasts, making them ill prepared to be used routinely in studying the cells of biological specimens.

Projection Tomography:

Projection tomography is the technique that makes a tomographic reconstruction algorithm by using projections of the specimen as its input data. There is an energy-dispersive detector that is sensitive to any signal that is produced along the column. 2D data is measured over a range of positions, measurements are made at several angles, and an analytic approach helps to construct a 3D map of the specimen. What distinguishes X-ray fluorescence micrographs from other tomographic methods is its self-absorption effects. This encompasses the re-absorption of the fluorescence by the specimen and the absorption of the incident beam. The self-absorption effects are increased for thick specimens when X-ray micrographs are done using low fluorescent energy.

Self-absorption has significant effects on XRF tomography. To combat this issue, good correction algorithms are required for image clarity they also will expand the specimen size domain, to lower fluorescence energies and maintain accurate data. Absorption maps can be used to estimate the self-absorption at various fluorescence energies. Using the energy-dispersive detector, inelastic and elastic signals were recorded. These inelastic and elastic signals provide access to major light element distributions that are normally hard to attain.

Confocal Tomography:

Confocal tomography is known as a direct-space approach to scanning the XRF tomography when axial resolutions are below 5 µm. In this approach, the signal derives from only a small portion of the illuminated column because the collimator confines the field view of the energy-dispersive direction.

Confocal tomography only gives a direct access to a small region of the specimen. Unfortunately, this can make it quite difficult to target the features of interest within a specimen. Researchers have suggested another approach to dealing with this issue: using projection tomography for a low-resolution overview and then following by using a confocal study of the region of interest.

Fluorescence imaging and cell function

One of the biggest goals of structural biology is to understand how cells function, and how they sense and process external and internal signals. Genetically encoded fluorescent proteins (FPs) and fluorescent sensors can help researchers visualize how cellular processes work. Cells, the basic building blocks of all living systems, rely on complex internal processes. These processes take place on the micrometer scale in different membrane compartments and cytoskeletal locations. Fluorescence imaging, employing green fluorescent protein (GFP) from the jellyfish Aequoria victoria and its relatives can help scientists further explore these cellular processes. GFP can be used as an important molecular imaging tool because of its fluorescence, or its capacity to produce a light of a different color other than the illuminating light. GFP and fluorescence can monitor cellular processes over time. Furthermore, GFP is encoded by a single portable DNA sequence that can be easily be fused to a protein of interest and expressed within living cells. Before GFP, researchers relied on fluorescent antibody techniques to examine the proteins and nucleotide sequences, but this could only be done on dead, fixed cells or tissue sections.

As GFP became a more and more popular molecular imaging tool, improvements and advances of GFP also arose. Mutagenesis of GFP caused an increase its brightness ad folding efficiencies and a decrease in its oligomerization. Mutagenesis also created forms of GFP that are photoactivable or photconvertable. It was discovered that GFP is just one member of a bigger family of homologous fluorescent proteins, mainly from marine corals, with different colors resulting from variations in structure and environment. Directed mutagenesis of the FP from these species resulted in a palette of FPs, covering the entire range of visible spectrum. Multiple colors allow for simultaneous imaging of multiple sets of proteins inside cells.

GFP developments ultimately gave rise to different imaging techniques such as fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, FRET, fluorescence cross correlation spectroscopy, total internal reflection microscopy, fluorescence lifetime imaging, and photoactivation localization microscopy (PALM). These imaging techniques allow for in vivo analyses of cell function.

For example FP reporters can be used to monitor the behavior of tagged signaling molecules and their organization as well as detect specific pools of each component in a signaling pathway. This can be done by photobleaching, where an area of the cell is photobleached with a high intensity laser pulse and the movement of unbleached molecules from neighboring areas into the bleached area is recorded by time-lapse microscopy. The kinetic properties of a protein within a cell, such as its movement between compartments, can also be seen when tagged with a genetically encoded FP. In both photobleaching and photoactivation, the overall functions of an FP fusion protein can be determined without disturbing other pathways or cell function.

Use of FPs in fluorescent imaging methods also enables protein-protein interactions to be resolved. This can be done through FRET measurements, which allow for mapping of protein-protein interactions within cells in real-time. In FRET, one reporter is a donor fluorophore, the other reporter is a longer wavelength acceptor fluorophore. The readout is energy transfer from the donor to acceptor. By incorporating GFP variants, these reporters can be attached to different proteins to test for their interaction.

Further processes developed include using probes for monitoring GTP hydrolysis and cell cycle events. One strategy is to use small molecules that can be induced to form dimers. Probes can be used to drive specific biological activities at selected times and places in cells. Another strategy involves optically inducible switches, which employ light to activate signaling molecules. Probes can also react with zinc and nitric oxide indicators, which are inorganic species that drive physiological processes or trigger pathology.

Reporter technologies allow for real-time visualization of biochemical processes in living systems and offer a means to obtain insights into spatial organization and regulation of intracellular signaling networks underlying biological processes.

Fluorescent Imaging of Kinase Action

Molecular imaging approaches have been developed in order to analyze the actions of phosphorylation in live cells. These involve the use of recently introduced fluorescent reporters that allow for high resolution imaging of phosphorylation in the cells. This molecular imaging provides us with understanding and insights of timing and cellular localization of signaling networks. Before the development of these fluorescent imaging techniques, measurements of kinase action was often done through analysis of enzymatic activities ex post facto (meaning retroactive) through techniques of immunoprecipitation or immunocytochemistry. The delayed measurements are not as effective as they have problems of specificity and the inability for reports on the kinase action in real time. In addition, obtainment of information after the kinase action can cause the loss of key information only found in live cells. As such, fluorescent reporters have been developed for the purpose of real time analysis in complex mixtures or living cells.

Protein Biosensors

Biosensors for peptides are often used to measure phosphoylation events with good resolution in vitro with the potetial for live cell imaging. These biosensors follow a basic design of synthetic flurophore into peptides or proteins. These properties change upon phosphorylation which allows for the analysis through a shift in wavelength (increases, decreases, or even both in quantum yield). There are four main types of bio sensors: environmentally sensitive, deep quench, self-reporting, or metal chelation enhanced.

Environmentally sensitive biosensors generally have a phosphospecific amino acid domain that complexes with the phosphoylated peptide. This type of biosensor is often used for Ser/Thr or Tyr phosphorylation. When activated, fluorescence is increased sevenfold.

Deep quench biosensors have a noncovalently attached quencher that shields the fluorescence until phosphorylation. When the molecule is phosphorylated the biosensors obtains a phosphospecific amino acid that separates the quencher from the fluorophore causing an increase in fluorescence. This often has around a 64-fold increases in fluorescence.

Self-reporting biosensors are used to detect tyrosine phosphorylation. For instance, tyrosine can be used to quench a fluorophore through pi-pi stacking interactions. When phosphorylated, the quench is lost and the fluorescence is increased by fivefold.

Lastly, metal chelation-enhanced biosensors use the nonnatural amino acid Sox, chelation-enhanced fluorophore and a series of biosensors for protein kinase activities that respond to Mg2+. These generally have a eightfold increase in fluorescence upon phosphorylation.

These biosensors are generally used in vitro kinase assays and allow for the detection of an initial lag phase. These biosensors also generally monitor activities of several kinases. There is also the possibility of using these biosensors to analyze live cell assays but these are more difficult. While these methods are good for analysis of the protein kinases, they are not as widely adopted as believed as they rely on specilizaed equipment and the instability of peptides and cellular perturbation can occur.

FRET-based Biosensors

FRET (stands for Forster (fluorescence) resonance energy transfer) is a mechanism to describe energy transfer between proteins. Most imaging techniques have the difficulty of getting the biosensor into living cells or have limitations that restrict use. FRET-based reporters generally overcome entering the cell as it has the cell manufacture the biosensor itself. These reporters are genetically encoded and can be transferred into cells as DNA. A notable example of these generically encoded biosensors is GFP (Green Flurorescent Protein). The changes in FRET can be seen as changes in emission rations between the fluorophore fluorescence; this is gernally the change in emission between donor and acceptor fluorophores.

FRET imaging is done through photochemical properties of donors using FLIM (Fluorescent lifetime imaging microscopy) which detects shortened fluorescence decay of the donor pair of FRET in the presence of the acceptor. This is advantageous as lifetime measurements are independent of the fluorophore concentration and photobleaching and allow for the distinguishing of actual FRET efficiency and probe concentration.

The FRET pair is selected to generate a maximum dynamic range. A domain is selected on the basis of the phosophoamino acid that is to be detected by the biosensor. The substrate part of the biosensor is what controls the specificity of the reporter. This is generally a short consensus peptide to be specifically recognied and efficiently phosphorylated by the target kinases but inert to other kinases. This method has given rise to a number of successfully genetically encoded biosensors. These have also been modified to include other features such as sequences to target a specific area of the cell or more genertion of the biosensors inside the cell.

These FRET based biosensors provide us with the means to image phosphoyrlation in living cells more efficiently and effectively than before.

Applications of Genetically Encoded Reporters

Genetically encoded reporters can provide sufficient sensitivity when compared to the fluoresence in peptide-based biosensors and are able to provide more useful information aboue the phosphorylation and regulation of signalling pathways. These reporters can be used to study specific kinases and influence activity. These can also target specific reporters to find correlations between phosphorylation of specific proteins to other features of the cells.

One of the more exciting applications of kinases reporters are through the usage of high throughput chemical screens. These can help pharmaceuticals as the reporters can ensure compounds achieve efficient cell entry and provide kinetic information of protein inhibition. This is a big change as these screens are generally done through in vitro and is unclear is these compounds can target the intracellular kinases. Through the usage of the in-cell kinases reporters, senveral novel PKA inhibitors were able to be identified. This was all done through the analysis of FRET.

References

• Piston, David. "Introduction to Fluorescent Proteins." 2008. http://www.microscopyu.com/articles/livecellimaging/fpintro.html. Dec 2, 2009.
• Lippincott-Schwartz, Jennifer. "Emerging In Vivo Analyses of Cell Function Using Fluorescence Imaging." Annual Review of Biochemistry. 2011.
• The New Genetics (2006): n. pag. U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES, National Institutes of Health, National Institute of General Medical Sciences. Web. <http://www.nigms.nih.gov>.
• Yellow fluorescent protein TagYFP. <http://www.evrogen.com/protein-descriptions/TagYFP-description.pdf>