A saposin-lipoprotein nanoparticle system for membrane proteins

Abstract

Membrane proteins are of outstanding importance in biology, drug discovery and vaccination. A common limiting factor in research and applications involving membrane proteins is the ability to solubilize and stabilize membrane proteins. Although detergents represent the major means for solubilizing membrane proteins, they are often associated with protein instability and poor applicability in structural and biophysical studies. Here, we present a novel lipoprotein nanoparticle system that allows for the reconstitution of membrane proteins into a lipid environment that is stabilized by a scaffold of Saposin proteins. We showcase the applicability of the method on two purified membrane protein complexes as well as the direct solubilization and nanoparticle-incorporation of a viral membrane protein complex from the virus membrane. We also demonstrate that this lipid nanoparticle methodology facilitates high-resolution structural studies of membrane proteins in a lipid environment by single-particle electron cryo-microscopy (cryo-EM) and allows for the stabilization of the HIV-envelope glycoprotein in a functional state.

Effects of partially dismantling the CD4 binding site glycan fence of HIV-1 Envelope glycoprotein trimers on neutralizing antibody induction

“…Our investigation of 293F VLPs was intended to eliminate this production bottleneck, but was unsuccessful, apparently due to contaminating material, possibly vesicles. An alternative platform to investigate membrane trimers that we are now investigating is to extract membrane trimers, then reconstitute them into liposomes or nanodiscs (Frauenfeld et al, 2016; Nakatani-Webster et al, 2015). In the meantime, the consistently positive nAb responses to N362Q mutant VLPs observed here should facilitate immediate efforts to formally re-evaluate the VLP dose required to induce nAbs, which could temporarily alleviate our current challenges in evaluating concepts with full concurrent controls (current doses are high in part to err on the side of caution).…”

Nanodiscs and solution NMR: preparation, application and challenges

“…This system is also compatible with a wide range of lipid molecules. Recently, Saposin-A discs were utilized for cryo-EM study of large membrane protein complexes like archaeal mechanosensitive channel T2 (32.9 kDa), with four predicted transmembrane helices existing as a putative homo pentamer, and bacterial peptide transporter PepT So2 (56 kDa), with 14 transmembrane helices existing as a homo tetramer [38]. …”

The potential use of single-particle electron microscopy as a tool for structure-based inhibitor design

“…New methodologies for extracting membrane proteins in more native states have been reported, for example styrene maleic acid lipid particles (SMALPs), which extract membrane proteins with their native lipids rather than in detergent micelles, and have been used for EM analysis (Postis et al, 2015;Lee et al, 2016). Furthermore, saposonin-lipoprotein nanoparticles have shown promise in high-resolution cryo-EM studies on membrane proteins (Frauenfeld et al, 2016). Nanodiscs also offer the potential to create more native environments that can be adapted for different lipid environments (Gao et al, 2016).…”