Researchers explore biosensor applications for fluorescence discovery

A coral protein with an intense blue colour has properties that could prove useful as a biological probe to track interactions between proteins and cells, according to new research from Monash and Queensland universities.

The Rtms5 pocilloporin protein, isolated from Great Barrier Reef coral species Montipora efforescens, is not itself fluorescent, but a single amino acid substitution creates a protein that fluoresces a deep red colour.

"Basically what we were looking for was a protein that had improved properties to what was available," said Monash researcher Dr Mark Prescott, who along with Dr Jamie Rossjohn and Assoc Prof Rod Devenish solved the structure of the protein in collaboration with the University of Queensland Centre for Marine Studies team led by Dr Sophie Dove and Prof Ove Hoegh-Guldberg that originally isolated the protein.

The crystallographic structure of the protein was recently published by the researchers in the journal Structure.

Commonly used fluorescent proteins include green fluorescent protein (GFP) originally isolated from jellyfish species Aequorea victoria, and the red fluorescing DsRed from Discosoma coral. The fluorescent proteins are all biochemically similar and all contain an identical three amino acid sequence that acts as a chromophore.

Rtms5 is shifted further into the red end of the absorption spectra, making it a potentially more useful protein, said Prescott.

"We were interested in finding new proteins that were red fluorescent but did not have the disadvantages of existing probes," Prescott said. "Proteins can fluoresce across the visible light spectrum, and we had been looking for proteins that emitted light further into the red part of the spectrum. Light emitted from such proteins is not as easily absorbed by mammalian tissue and is easier to detect."

But the wild-type protein does not fluoresce, and the Monash researchers were interested in finding out why. Using protein crystallographic techniques to examine the structure of Rtms5 and its fluorescent variant, some interesting properties of the protein were discovered. Compared to the equivalent structure in GFP and DsRed, the orientation of the chromophore was flipped around.

"We think the reason this protein is non-fluorescent is because it has a unique orientation of its chromophore -- the structure that absorbs light -- that has not been seen before in any other protein," said Prescott.

Deeper into the red

However, the chromophore orientation is not the only factor at play, he said. The fluorescent variant of Rtms5, which has a single amino acid change from histidine to serine at position 146, has a 170-fold increase in fluorescence, despite an apparent lack of large-scale structural changes such as an alteration in the chromophore orientation.

"We have found we can manipulate the protein to make it fluoresce a deep red colour," Prescott said. The researchers are continuing to further elucidate and define the structural mechanisms responsible for fluorescence.

The researchers are also using the structural knowledge they have acquired to design and make better probes that fluoresce further into the red than DsRed. The fluorescent protein is already being used by Monash scientists to monitor proteins in cells.

The protein has another interesting feature that intrigues Prescott - it changes colour depending on the pH, from the deep blue colour seen at an alkaline pH to a yellow colour under acidic conditions. The pH sensitivity may be of use in developing a pH biosensor, said Prescott.

"The thing about these proteins is that they all have interesting properties... which point to interesting applications," he said. "Its unique properties could make it an extremely useful scientific tool."