*4 Year MRC PhD Programme* Development of a microscopy-based method for monitoring domain flexibility of macromolecular complexes in living cells

The biological activities of macromolecular complexes involve dynamic movements of their domains or subunits, but the kinetics of such motions in the cellular environment are mostly unknown due the lack of suitable approaches. This project aims to develop a microscopy-based method that monitors changes in domain flexibility in real time in live cells. The method being developed will utilize the ability of a flexible segment between a fluorophore and a component of a protein complex to increase the rotational diffusion of the fluorophore relative to its rigid attachment to the same complex. We will assess such rotational diffusion using time-resolved fluorescence anisotropy (TRFA) [1], as readout for flexibility of a fluorophore-flanked domain.



As a biological system, we will use a protein complex known as the multi-subunit Cullin3 (Cul3)-Keap1 E3 ligase complex. Under normal conditions the principal substrate of this complex, Nrf2 – which itself controls the cellular redox homeostasis – is rapidly degraded following Cul3-Keap1 dependent ubiquitination [2]. However, oxidative stress modifies Keap1, the sensor protein within the E3 ligase complex, leading to inactivation of the E3 ligase, stabilization of Nrf2 and the induction of antioxidant responses. Activation of Nrf2 is protective in many animal models of chronic disease and Nrf2 in now considered a drug target. Detailed understanding of the interaction of Nrf2 with the multi-subunit Cul3-Keap1 E3 ligase complex is therefore essential for targeting Nrf2 for disease prevention and treatment.



Nrf2 is recruited to dimeric Keap1 via two unequal Keap1-binding sites at its N-terminus, and the transitions between 1-site and 2-sites bound states are critical for regulation of Nrf2 degradation. Using this method, we aim to distinguish 1-site and 2-sites bound states of Nrf2 in cells based on the difference in flexibility of the N-terminus of Nrf2, visualized by anisotropy decays of a long-fluorescent lifetime tag [3] linked to the N-terminus of Nrf2, in vitro and in live cells. This project sits at the interface between biology and physics and will provide training in cloning, recombinant protein expression and purification, cell culture, and quantitative fluorescence microscopy.