Altering the oligomerization state of GFP-like purple protein to enhance protein taggin ability

Since its discovery and purification in the 1960s, Green Fluorescent Protein has quickly become an extremely useful scientific tool for analyzing protein expression and dynamics. GFP was originally discovered in a species of jellyfish, Aequorea victoria, apparent only in response to a blue flash of bioluminescence. Upon examining its structure, scientists soon found that the source of GFP’s color was an autocatalytically formed chromophore at the center of its β-barrel structure. Because of this autocatalytic mechanism of fluorescence, the GFP gene alone can be attached to proteins of interest and used to track expression and movement in vivo. While GFP itself was a breakthrough in the biotechnology world, it is hardly the end of the line as far as fluorescent proteins go. In recent years, scientists have been searching for and finding more members of the GFP-family that have the same autocatalytic property but display different colors. The introduction of new colors to the library of fluorescent proteins offers great advantages in multiple protein tagging and studying protein interactions. However, most fluorescent proteins that have been found to date have been tetramers, consisting of four identical subunits. This quaternary structure makes them ineffective as protein tags. Researchers have recently been exploring ways to alter the oligomer ization state of fluorescent proteins and create dimeric and monomeric forms the can be useable tags. In my experiment, I attempted to do so in a GFP family protein from species Galaxea fascicularis and Montipora efflorescens known as purple protein. There is not a protein tag that emits is colored purple currently, and so my goal was to reduce the oligomerization state of purple protein, assuming that it is a tetramer, while maintaining its purple color. To do this, I used site-directed mutagenesis techniques to change specific amino acids in the protein that were predicted to be involved in formation of tetramers. All three mutations made in the sequence affected the spectral qualities, as the cells they that the mutants were transformed into showed no purple expression. One mutation resulted in a protein that migrated at half the relative molecular weight of the wild type protein in a gel filtration column, and thus we were successful in reducing the oligomerization state of the purple protein through that mutation. While it is not currently useful as tag, the successful mutant should be used in future experiments to isolate a functional monomer of the purple protein, adding to the library of protein tag colors.