Biophysical characterization of nanodiscs and nanodisc-reconstituted mechanosensitive ion channels

Abstract

Lipid-protein interactions are crucial for life. Research into this area is continuously growing, not only in the context of basic science, but also from a technological perspective aiming to facilitate their handling and stability for further studies. The development of nanodisc (ND) technology, with apolipoprotein-A1 derived membrane scaffold protein (MSP), has become key to the study of lipid-protein complexes, including mechanosensitive (MS) proteins. Although it is a widely used artificial lipid environment for membrane proteins in vitro, how ND structures assembly together remains poorly understood. In this thesis, I apply ensemble and single-molecule Förster Resonance Energy Transfer (FRET) assays to investigate the dynamics of ND structures and ND-reconstituted MS channels. First, I describe a FRET-based characterization of NDs. My studies reveal the stepwise mechanism of ND assembly and disassembly, the timescales of these processes and the stability of ND under detergents with or without the membrane protein. Next, I use this MSP-ND technology to investigate the structure of the pentameric MS channel of large conductance (MscL), which has attracted considerable attention over the past years. MS channels allow cells to sense and response to external mechanical stimuli. However, most studies have used ensemble measurements providing only average-conformation values. Here, I present a library of FRET-labelled MscL proteins to characterize the conformation of MscL channels at single-molecule level. Lastly, completely understanding mechanosensation requires to manipulate membrane tension and simultaneously image their response one by one. In the last part of this thesis, I demonstrate the feasibility of incorporating magnetic beads inside Giant Unilamellar Vesicles (GUVs) as a step towards the future development of a combined FRET-magnetic tweezers to explore mechanosensation under varying tension. Overall, this project expands our knowledge of NDs and explores potential methods to study single MS channels by fluorescence-force hybrid microscopy.