Microfluidic diffusional sizing probes lipid nanodiscs formation

The biophysical characterisation of membrane proteins and their interactions with lipids in native membrane habitat remains a major challenge. Indeed, traditional solubilisation procedures with detergents often causes the loss of native lipids surrounding membrane proteins, which ultimately impacts structural and functional properties. Recently, copolymer-based nanodiscs have emerged as a highly promising tool, thanks to their unique ability of solubilising membrane proteins directly from native membranes, in the shape of discoidal patches of lipid bilayers. While this methodology finally set us free from the use of detergents, some limitations are however associated with the use of such copolymers. Among them, one can cite the tedious control of the nanodiscs size, their instability in basic pH and in the presence of divalent cations. In this respect, many variants of the widely used Styrene Maleic Acid (SMA) copolymer have been developed to specifically address those limitations. With the multiplication of new SMA copolymer variants and the growing interest in copolymer-based nanodiscs for the characterisation of membrane proteins, there is a need to better understand and control their formation. Among the techniques used to characterise the solubilisation of lipid bilayer by amphipathic molecules, cryo-TEM, ³¹P NMR, DLS, ITC and fluorescence spectroscopy are the most widely used, with a consensus made in the sense that a combination of these techniques is required. In this work, we propose to evaluate the capacity of Microfluidic Diffusional Sizing (MDS) as a new method to follow copolymer nanodiscs formation. Originally designed to determine protein size through laminar flow diffusion, we present a novel application along with a protocol development to observe nanodiscs formation by MDS. We show that MDS allows to precisely measure the size of nanodiscs, and to determine the copolymer/lipid ratio at the onset of solubilisation. Finally, we use MDS to characterise peptide/nanodisc interaction. The technique shows a promising ability to highlight the pivotal role of lipids in promoting interactions through a case study with an aggregating peptide. This confirmed the relevance of using the MDS and nanodiscs as biomimetic models for such investigations.