Exploring “hidden” fluorescent protein photophysics and energy transfer dynamics

Genetically encodable fluorescent proteins (FPs) have revolutionised biophysics, allowing the direct visualisation of structures and processes in living cells. More recently their ability to report molecular level interactions through Forster resonance energy transfer (FRET) has become feasible. In FRET, electronic excitation is transferred once a critical proximity radius (ca 5 nm in FPs) has been reached, which allows protein-protein interactions and conformational changes to be explored. FRET is most cleanly detected through lifetime shortening of the FP donor population. However, a largely unrecognised feature of many FPs is the presence of multiple exponential fluorescence decays, often accompanied by different transition dipole orientations. The critical FRET radius strongly depends on transition dipole orientations, and whilst one FP pair may undergo FRET, another at the same distance will not. This heterogeneity severely complicates things. This project aims to unravel its origins and will probe fundamental and largely unexplored FP photophysics and FRET with a battery of state-of-the art techniques. Broadly tuneable femtosecond laser systems for single and two-photon excitation will be combined with fast time resolution correlated single photon counting (TCSPC) and single molecule fluorescence lifetime techniques.

In collaboration with researchers at the NIH (Bethesda Md and Montana State University) and UC San Diego we will explore existing and newly developed FPs and investigate FRET dynamics between FP pairs and in multiple donor-acceptor systems where anomalous surplus energy transfer effects arising from quantum entanglement may be involved. The project is part of a successful (15-year) collaboration between Angus Bain (Physics & Astronomy) and Tom Blacker (Structural & Molecular Biology) and will involve work in both laboratories. Prior expertise in the techniques used in either lab is not a prerequisite as full training will be given. A strong interest in pursuing interdisciplinary research and exploring unanswered questions is a definite advantage.