Abstract
SummarySaposin-derived lipid nanoparticles (SapNPs) are a new alternative tool for membrane protein reconstitution. Here we demonstrate the potential and advantages of SapNPs. We show that SapA has the lowest lipid specificity for SapNP formation. These nanoparticles are modular and offer a tunable range of size and composition depending on the stoichiometric ratio of lipid and saposin components. They are stable and exhibit features typical of lipid-bilayer systems. Our data suggest that SapNPs are versatile and can adapt to membrane proteins of various sizes and architectures. Using SapA and various types of lipids we could reconstitute membrane proteins of different transmembrane cross-sectional areas (from 14 to 56 transmembrane α helices). SapNP-reconstituted proteins bound their respective ligands and were more heat stable compared with the detergent-solubilized form. Moreover, SapNPs encircle membrane proteins in a compact way, allowing structural investigations of small membrane proteins in a detergent-free environment using small-angle X-ray scattering.
Highlights
Membrane proteins (MPs) are encoded by 20%–30% of open reading frames in sequenced genomes (Wallin and von Heijne, 1998) and provide key functions for inter- and intracellular communication
We show that SapA has the lowest lipid specificity for saposin-derived lipid nanoparticles (SapNPs) formation
Our data suggest that SapNPs are versatile and can adapt to membrane proteins of various sizes and architectures
Summary
Membrane proteins (MPs) are encoded by 20%–30% of open reading frames in sequenced genomes (Wallin and von Heijne, 1998) and provide key functions for inter- and intracellular communication. Isolating and studying MPs in aqueous buffers are notoriously difficult tasks and require the use of substituting molecules to compensate for the nursing hydrophobic acyl chains of the lipids and to prevent aggregation To date, this is mainly achieved by the use of detergents (Duquesne and Strugis, 2010), which, by definition, are amphipathic surfactants able to solubilize lipids. In an attempt to overcome the hurdles associated with the use of detergent, various alternative surfactant molecules or polymers have been developed Such tools include fluorinated surfactants (FSs) and amphipols (Breyton et al, 2010), both of which were shown to increase the biochemical and physical stability of MPs, compared with detergents (Kleinschmidt and Popot, 2014; Abla et al, 2015), and have been successfully used in structural studies including smallangle neutron scattering (Breyton et al, 2013a, 2013b) and single-particle electron microscopy (Liao et al, 2013; Beckham et al, 2017).
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