Characterisation of an orange chromoprotein and its application in pcFRET.

2017-02-16T23:45:09Z (GMT) by Don Paul, Craig Bradley
The GFP-superfamily of proteins represents an indispensable tool for use in molecular cell biology research. The last two decades have witnessed many improvements and innovations in the technology. Many of the developments in the field were driven by an increased understanding of the intricate interactions at play within these proteins and thus, it is now common practice to determine the 3D structure of new variants. The availability of new fluorescent proteins with different but complimentary excitation and emission spectra suitable for Förster resonance energy transfer (FRET) promoted their application for monitoring protein-protein interactions, and as biosensors for following events in live cells. The work undertaken during the course of my PhD studies focused on the characterisation and application of a novel weakly fluorescent photochromic protein called Phanta. My initial work focused on the characterisation of this protein as a ‘dark’ pcFRET acceptor. Later experiments involved a detailed study of both the optical and structural properties of Phanta and related variants with the aim of gaining useful insight into its unique properties. Weakly fluorescent proteins might be considered by most researchers to be of little use for their experiments, and Phanta (and other similar proteins) might have been discarded due to its poor fluorescence emission. My work suggests that these proteins, when used in combination with suitable fluorescent donor proteins, will have a bright future. Researchers are continually looking for options to increase the number of events that can be monitored in biological systems, and my work highlights the application of Phanta in the novel imaging technique of pcFRET.