Masters, T.A.; (2009) Time-resolved fluorescence studies of enhanced green fluorescent protein and the molecular dynamics of 3- Phosphoinositide Dependent Protein Kinase 1. Doctoral thesis , UCL (University College London).

Abstract

Fluorescent proteins (FPs), particularly Enhanced Green Fluorescent Protein (EGFP), are essential tools in the study of intact biological systems. Whilst the photophysics of its progenitor, GFP, have been investigated extensively, far fewer studies of EGFP have been made. In this thesis, a full characterisation of EGFP excited state photophysics by singleand two-photon time-resolved fluorescence lifetime and anisotropy is presented. Furthermore, the two-photon transition tensor, determined by absorption and initial anisotropies, is shown to be dominated by a single element. The two-photon excited state of EGFP was subject to Stimulated Emission Depletion (STED), revealing the stimulated emission cross section, the ground state relaxation time and the time evolution of the higher order distribution moments to which anisotropy is not sensitive. The strong adherence to theoretical Debye diffusion reinforced the conclusions of the two-photon structure model, and showed EGFP to be an excellent molecule for the future development of STED. In addition, these studies provided a sound basis on which to employ single- and two-photon FRET in vivo and in vitro. Cell behaviour is governed by the transduction of molecular signals from the extracellular environment to intracellular compartments. At the centre of the PI 3-kinase signalling pathway is PDK1, a Serine/Threonine kinase, which phosphorylates numerous important downstream targets including Protein Kinase B (PKB). To date however, the regulatory mechanisms governing the behaviour of this protein remain poorly understood. Timeresolved fluorescence lifetime imaging microscopy (FLIM) was employed with FP tagged PDK1 to investigate dynamic interactions in intact cells in situ and in vivo. PDK1 was shown to dimerise in a manner dependent on PI 3-kinase activity and PDK1 PH domain lipid binding. To detail the structure of the observed intermolecular interaction, recombinant FP labelled PDK1 was produced with insect cells. Measurement of the rise in acceptor fluorescence during FRET in vitro indicated the PDK1 dimer pair exists in an antiparallel arrangement. These results provide the first insight on the structure of the dimer and demonstrate that the generation of 3-phosphorylated lipids is required for its formation.

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