Scientists have recently reported a new fluorescent protein which has the makings of an ideal fluorescent tag. Light is essential for visibility. But light rays can’t penetrate the skin; this makes visualisation of a cell and its components difficult. The discovery of fluorescent proteins was like switching on the proverbial bulb. Scientists were able to trace entire metabolic pathways by tagging cells with these fluorescent markers. Green fluorescent protein (GFP) was the first fluorescent protein to be discovered and it remains the most widely used fluorescent marker till date. But it has its share of limitations.
Recently, S Ramaswamy from the Institute for Stem Cell Biology and Regenerative Medicine (inStem), Bangalore and his colleagues from National Center for Biological Sciences (NCBS), Bangalore, the University of Wisconsin, USA, and University of Iowa, USA have isolated a fluorescent marker from a blue coloured morph of Walleye (Sander vitreous) — a popular sports fish native to North America. Both blue and yellow Walleyes have been described in the literature but the information on the nature of these pigments was lacking. The blue colour was thought to result from some copper-containing compound or cyanobacterium present on the fish mucus. Ramaswamy and his colleagues were looking for the elusive bacteria and the blue pigment. But after several failed attempts at isolating the bacteria, they realised that the blue pigment could be encoded in the fish genome. Whole genome sequence studies ensued, leading to the discovery of the protein which was named Sandercyanin, after the fish (Sander vitreous) and the blue color (cyan).
The isolated protein presented interesting properties — it shows bright red fluorescence when illuminated with UV radiation. “Red fluorescence ensures high signal strength and low background scattering. This would prevent the mixing of excitation and emission wavelengths and simplify microscopy experiments,” says Swagatha Ghosh, a graduate student in Ramaswamy’s lab. “Most fluorescent proteins suffer photobleaching when exposed to UV light for extended time periods due to disruption of bonds and damage to structure. But Sandercyanin is extremely photostable and doesn’t bleach over time.” she adds.
The red fluorescent protein has huge potential in deep tissue visualisation. But this requires expression of Sandercyanin within the cells. The scientists therefore sequenced and packaged the Sandercyanin gene into E. coli genome for recombinant expression. The protein was extracted, crystallised and compared with the native protein originally isolated from blue walleye for structural integrity. The purified protein, though colorless, transforms to a brilliant blue shade when mixed with biliverdin, a pigment that is produced when UV radiation degrades heme. Scientists have traced this phenomenon to the formation of protein tetramers in the presence of biliverdin which is essential for fluorescence. The role of biliverdin also serves to demystify the seasonal variation of blue walleye sightings.
The frequency of finding blue walleyes increases in summer. Out on a fishing expedition, Wayne Schaefer, also an author on this study, realised that increased biliverdin may be produced due to greater UV exposure in summer. While this waste product is being removed from the body through the skin, it can get trapped in the mucous membranes and associate with Sandercyanin giving it a characteristic blue colour. Sandercyanin is also found in traditional yellow walleyes. But it is only in the presence of biliverdin that they assume a blue coloured tetramer conformation. This adduct can then absorb higher energy damaging UV rays and re-emit them as low energy red fluorescence. “In this sense, presence of Sandercyanin on walleye skin is a remarkable example of nature protecting itself by using the very product formed as a result of damage (biliverdin),” says Schaefer.
The scientists already hold one patent for the identification and development of Sandercyanin as a fluorescent tag. The results of this study have enabled them to identify specific amino acids involved in oligomerisation of the protein and the mechanism that causes biliverdin associated Sandercyanin to fluoresce. Armed with his information they hope to engineer monomeric Sandercyanin variants with high quantum efficiency. The smallest red fluorescent protein identified till date, and the first from a vertebrate, Sandercyanin opens up new possibilities in imaging.