Difference between revisions of "Team:Uppsala/Project/UnaG"

Go Green - Go UnaG!

As part of this iGEM project, research has been done on the newly discovered green fluorescent protein called UnaG. This protein is interesting firstly because it is about half the size of other fluorescent proteins that are being used today, making it suitable as a fusion protein. Secondly, it fluoresces only when a bilirubin molecule is bound non-covalently to the protein. This feature could make UnaG suitable as an inducible marker by the addition and removal of bilirubin to cells. Moreover, unlike all other fluorescent proteins of today, UnaG does not need molecular oxygen in order to fluoresce, meaning that it can be used as a marker protein in research under hypoxic conditions. These cool features, alongside the fact that it is always exciting to work with new discoveries, made us want to add this protein to the iGEM toolbox! During the summer we created three different UnaG biobricks; one with a hexahistidine tag suitable for affinity chromatography, one with the hexahistidine tag and a flexible linker of six amino acids suitable for fusing to other proteins, and lastly one biobrick with only the flexible linker attached to UnaG.

UnaG - A Fluorescent Member of the FABP Family

UnaG was discovered as late as 2013. It originates from the muscle fibers of the Japanese Unagi eel Anguilla japonica. This is the first fluorescent protein to be found in a vertebrate species, giving it a range of biochemical properties that differs from proteins found in the original FP family - one of the most astounding ones being that fluorescence occurs only when a ligand binds non-covalently to the protein, namely bilirubin. (I)

Structure of UnaG

UnaG is a fatty acid-binding protein (FABP). These proteins are cytosolic and exist in most vertebrates and invertebrates. They contribute to the transport of lipophilic compounds, often fatty acids. Although it is still rather evasive in which ways FABPs function in cells, some of these proteins appear to play roles in cellular anabolism. Wild-type UnaG is 139 amino acids long and has a molecular weight of 15.6 kDa. UnaG is a ß barrel structure containing eleven ß strands - a typical quality for FABPs. Instead of binding a fatty acid molecule, the ligand of UnaG is bilirubin, which binds non-covalently to the center of this ß barrel. Bilirubin exists in all vertebrates and is a metabolite of the heme group in hemoglobin. Bilirubin can only bind to UnaG in its unconjugated (4Z,15Z) form, meaning that wild-type UnaG shows high specificity to this particular molecule. In fact, BR is the actual fluorophore of UnaG. When BR binds to the cavity of the protein, it emits green light at 527 nm. Holo-UnaG has maximum absorption at 498 (i.e. blue light) nm. UnaG does not need molecular oxygen in order to fluoresce, making it suitable for research executed under anaerobic conditions where FPs are not functional. According to Akiko Kumagai and his team, the quantum yield of holo-UnaG is 0.51 and it is stable in a pH ranging from 4 to 11 (I). GFPs are usually stable in a pH range from 6-10, but this varies depending on which mutant is used. (II)

Using UnaG as a Fluorescent Marker

Apo-UnaG does not fluoresce, but Akiko Kumagai’s research group showed that Holo-UnaG (i.e. UnaG+BR) fluoresces instantaneously. No other fluorescent protein has a ligand that binds non-covalently to it. This could mean that it is possible to create an inducible fluorescent marker that could be used instead of photoswitchable FPs. Bilirubin does not exist naturally in lower classes of organisms such as bacteria and yeast, so it would have to be added exogenously when research is being done on these types of cells. UnaG has shown the ability of being fused to other proteins via a flexible linker both on the C terminus and the N terminus, making it suitable to use as a fusion marker. (I)

Since UnaG was discovered fairly recently, no mutagenic variations of the protein have been developed with improved characteristics than those of wtUnaG. However, several fluorescent FABPs have recently been discovered in other marine organisms. Two of them are called Chlopsid FPI and FPII. These two were also found in eels, although in another species, and both of them emit green fluorescence. Researchers have identified a tripeptide motif – Gly-Pro-Pro – conserved in all fluorescent FABPs. Chlopsid FPI and FPII are also bilirubin inducible. Alignment of the DNA sequences of UnaG and the Chlopsid proteins show 54% homology. There is a slight blue shift in the fluorescence (UnaG is 498/527 nm ex/em and Chlopsid FPI and FPII 489/523 nm ex/em). The researchers suggest that this blue shift stems from the fact that the 57th amino acid in UnaG is asparagine, whereas Chlopsid FPI and FPII contain a histidine at this site. The histidine would contribute to a more π-conjugated system, resulting in a difference in fluorescence. This discovery suggests possibilities of changing the emission wavelength of these proteins by mutagenesis comparableto what has been done on GFP for the past 40 years. This could in that case enable creation of fluorescent FABPs of different colors. (III)

References

Bilirubin

Bilirubin is a yellow pigment (Benioff Children’s Hospital, San Francisco) created by the enzymatic reaction of haemoglobin degradation. Since the haemoglobin is located inside the red blood cells, the protein is hidden from the catabolism. It is only when the exchange into new red blood cells takes its place that the haemoglobin becomes exposed and decomposed by either fagocytosis or haemolysis. The result of the degradation are two toxic compounds; carbon monoxide (CO) and the bilirubin, were as it is only the carbon monoxide that is possible for the body to excrete easily (Mugnoli et al. 1983). The bilirubin is difficult to excrete since it is a non-polar compound and therefor not soluble in water (Fevery, J et al. 1977). The bilirubin molecules have a tendency to create different complexes with other proteins as well, such as serum albumin. The pigment also has the ability to conjugate with three types of proteins that are involved in the transport and the metabolism of bilirubin in vivo (Bonnett et al. 1978).

Bilirubin is an unstable compound (Le Bas, Allegret et al. 1980) and can occur both as bound and free pigment among the humans. The molecule has a tendency to form several derivate of bilirubin such as biliproteins, several conjugates and glucuronides etc (Harris, Kellermeyer, 1970). The schematic form of bilirubin (Figure 1B.) is almost never present (if so the molecule would have been directly polar and lipophilic) (Mugnoli et al. 1983). The more stable and dominant formation of bilirubin (Figure 1C.) is present when bilirubin is in liquid and solid constellations (Le Bas, Allegret, et al. 1980). In these forms the pigment creates several hydrogen bonds between the NH/O-groups and OH/-groups, which in turn makes the hydrophilic COOH- and NH-groups not available for polar interaction with their environment. Consequently the bilirubin becomes insoluble in water and methanol, but easy to dissolve in chloroform (Mugnoli et al. 1983). Even at the pH-values where the COOH-group is in an ionic state the formation of Figure 1C is predominant. These features also occur in well-known lipophilic compounds such as dioxins and polychlorated biphenyls (PCBs). Common properties for these compounds, among which bilirubin is included, is that they can diffuse through biological membranes in the placenta, blood brain barrier and hepatocytes.

Figure 1B: Linear structure of bilirubin. (Antony F. Mcdonagh et al. 1985). Figure 1C: Crystalline and liquid structure of bilirubin. (Antony F. Mcdonagh et al. 1985).

Bilirubin is naturally excreted in the liver by, with the help of enzymes, conjugating with the sugar glucoronic acid to create glucuronides. Since the glucoronic acid contains several COOH- and OH-groups the bilirubin conjugate becomes more hydrophilic than the bilirubin alone. Thereby it becomes possible for the bilirubin to leave the liver and go along with the bile, through the kidneys and then finally get excreted with the urine.

One chain of glucuronic acid is enough to make the bilirubin excretable, but diglucoronides can also occur in the human bile. These contains two glucuronic acids per bilirubin molecule and the compound seems to simply be an effect of bilirubin’s three dimensional structure. No indications that the diglucuronides are more biological efficient have been noticed so far. The human being´s survival is dependent on the monoglucuronides, but not on the diglucuronides (Gordon et al. 1976).

Hyperbilirubinemia, also called Jaundice disease or icterus (www.mesh.kib.ki.se), occurs when either the formation of bilirubin glucuronides or the excretion of these does not work in the correct manner. Consequently the concentration of bilirubin and glucuronic acid accumulates in the body (Buchan, 1826). High levels of bilirubin are toxic and it can result in neurologic harm that causes deafness, delayed development and cerebral pares (Benioff Children’s Hospital, San Francisco).

References

Literature

Results

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