The development and application of real-time protein interaction technology

Protein interactions are a fundamental part of cellular processes, and represent a key target in the understanding of cell behaviour, communication and function. These interactions are dynamic in nature, and change over time. Furthermore, interactions can be dependent on co-localisation in cellular compartments. Both of these characteristics can be obscured through observation by bulk molecular cell assays such as co-immunoprecipitation (co-IP) and can obscure the intricacies of single cell dynamics. The development of tools and new methodologies to observe single cell protein interaction dynamics are key to understanding the underlying mechanisms that direct cell fate. Systems biology aims to incorporate into predictive models of the whole system. In this way, the development of quantitative experimental tools is a key component of systems biology. Forster Resonance Energy Transfer is a widely used technique in the field of protein-protein interaction studies. Dependent on the non-radiative transfer of energy from a fluorescent molecule of higher excitation energy to a fluorescent molecule of lower excitation energy with sufficiently overlapping spectra, the process occurs across a 1-10nm (100A) range. As a result, FRET interactions between fluorophores attached to biologically functional proteins are a strong indication of protein interaction. The photoswitchable protein Dronpa offers a unique opportunity to develop a real-time live cell variant of this technique. Through the modulation of Dronpa fluorescence, repeated donor quenching can be observed, allowing quantification of FRET interactions between fluorophores without spillover. This is achieved sequentially through the optimisation of Dronpa imaging parameters for live cell imaging, followed by the identification and testing of candidate FRET partners using an optimised imaging protocol. Direct fusions were used to qualify potential FRET responses between tested pairs of fluorophores. FRET responses using positive control constructs were rigorously tested and quantified to ensure repeatable and consistent reporting of FRET. The spectral properties of Dronpa and chosen FRET partners were also rigorously tested to ensure FRET could be accurately measured through sensitised emission. Following the confirmation of a reliable FRET response using positive control constructs, the assay was applied to the NF-κB protein p105, and other interacting family members to measure any changes in protein dynamics. The system was applied both on a single switch basis to make comparisons between different combinations of co-expressed fusion proteins and, in time series experiments, to measure potential changes in dynamics over time. p105 showed interesting behaviour not reported in the literature, specifically the detection of full length p105 in the nucleus before stimulation and the strong intramolecular interaction of N and C terminal regions which become perturbed by co-expression of p65. p65 displayed a stronger interaction with the c-terminal region of p105, indicating preferential binding of p65 with ankyrin repeat region rather than the Rel homology domain of p105. Fluorescence Cross Correlation Spectroscopy (FCCS) was used as a complimentary technique to confirm findings in the FRET assay. Considered together, these data suggest Dronpa is a viable component for the accurate detection of FRET in real time, free from complication by spillover. The use of this technique could help to elucidate dynamic protein interactions key to the development and improvement of existing models in systems biology approaches.