Fluorescence resonance energy transfer studies of protein interactions

Abstract

This thesis presents an investigation of fluorescence resonance energy transfer (FRET) as a reporting signal for protein-protein interactions. Quantitative optical assays to measure protein binding, conjugation and deconjugation are developed and results validated by conventional biochemical techniques. The optical techniques developed provide fast, cheap, quantitative and accurate alternatives to conventional methods. Fluorescent protein fluorophores ECFP and Venus-EYFP were chosen as they are a non-interfering FRET pair and provide an inexpensive and convenient cloning-based labelling method.

The small ubiquitin-like modifier SUMO and the SUMOylation pathway leading to its conjugation to target proteins is investigated as a model system. These assays are hence particularly relevant to research on post-translational modification and ubiquitin systems. In protein-protein binding assays we utilise both steady-state and time-resolved FRET detection to measure the equilibrium binding constant of the well-characterised pair SUMO1 and Ubc9. An assay in multi-well plate format is also presented, which uniquely enables repeat measurements under varying conditions and under the addition of further substances. The multi-protein binding interactions of the SUMOylation pathway including RanBP2 are analysed in binding inhibition assays. Our results clarify the role of RanBP2: a covalent SUMO1-Ubc9 link is required for the formation of a trimeric complex, although mutual binding sites are present on all three proteins. Furthermore, the binding of SUMO1 and Ubc9 is disrupted by RanBP2, which may be an essential step in transferring SUMO1 to its target protein. A FRET-based kinetic study of this conjugation process to RanGAP1 is presented. An assay to monitor the deconjugation of SUMO1 by specific proteases is established using a doubly-tagged SUMO construct. This enables a quantitative analysis of protease and substrate specificity based on real-time kinetic data, a characterisation of crude cell extracts and a high-throughput screen for protease inhibitors using FRET. A screen of the National Cancer Institute (NIC) diversity set for SenP1 inhibition reveals nine suitable compounds, which are potential anti-cancer drugs.

The results of two further projects, the study of protein-protein binding by measuring small refractive index changes and the autofluorescence of normal and neoplastic cervical tissue models are also presented. In the latter, principal component analysis was used to systematically identify emission regions of significant variation between samples, enabling discrimination between healthy and pre-cancerous tissue models.