Preparation and Characterization of Stable -Synuclein Lipoprotein Particles

Multiple neurodegenerative diseases are caused by the aggregation of the human -Synuclein (-Syn) protein. -Syn possesses high structural plasticity and the capability of interacting with membranes. Both features are not only essential for its physiological function but also play a role in the aggregation process. Recently it has been proposed that -Syn is able to form lipid-protein particles reminiscent of high-density lipoproteins. Here, we present a method to obtain a stable and homogeneous population of nanometer-sized particles composed of -Syn and anionic phospholipids. These particles are called -Syn lipoprotein (nano)particles to indicate their relationship to high-density lipoproteins formed by human apolipoproteins in vivo and of in vitro self-assembling phospholipid bilayer nanodiscs. Structural investigations of the -Syn lipoprotein particles by circular dichroism (CD) and magic angle solid-state nuclear magnetic resonance (MAS SS-NMR) spectroscopy establish that -Syn adopts a helical secondary structure within these particles. Based on cryo-electron microscopy (cryo-EM) and dynamic light scattering (DLS) -Syn lipoprotein particles have a defined size with a diameter of approximate to 23 nm. Chemical cross-linking in combination with solution-state NMR and multiangle static light scattering (MALS) of -Syn particles reveal a high-order protein-lipid entity composed of approximate to 8-10 -Syn molecules. The close resemblance in size between cross-linked in vitro-derived -Syn lipoprotein particles and a cross-linked species of endogenous -Syn from SH-SY5Y human neuroblastoma cells indicates a potential functional relevance of -Syn lipoprotein nanoparticles.