Abstract
Based on the saposin-A (SapA) scaffold protein, we demonstrate the suitability of a size-adaptable phospholipid membrane-mimetic system for solution NMR studies of membrane proteins (MPs) under close-to-native conditions. The Salipro nanoparticle size can be tuned over a wide pH range by adjusting the saposin-to-lipid stoichiometry, enabling maintenance of sufficiently high amounts of phospholipid in the Salipro nanoparticle to mimic a realistic membrane environment while controlling the overall size to enable solution NMR for a range of MPs. Three representative MPs, including one G-protein-coupled receptor, were successfully incorporated into SapA-dimyristoylphosphatidylcholine nanoparticles and studied by solution NMR spectroscopy.
Highlights
A round 30% of all genes encode integral membrane proteins (MPs),[1] which have a range of important biological functions, leading to their involvement in many diseases
The disc-like particles consist of a phospholipid bilayer enclosed by two beltlike membrane-scaffold proteins (MSPs),[10] with the MP of interest incorporated into the center of the discs
This idea was confirmed previously in a cryo-electron microscopy (EM) study, where an archaeal mechanosensitive channel T2 (32.9 kDa), which is a putative homopentamer, and a bacterial homotetramer peptide transporter PepTSo2 (56 kDa) were both incorporated into Salipro nanoparticles at pH 7.4.22 This study demonstrated that Salipro
Summary
Communication nanoparticles show promising flexibility, enabling incorporation of a range of protein sizes without the need to screen multiple different scaffold−protein constructs. A chemical-shift-based comparison of OmpX in Salipro[3], with the same protein embedded in ΔH5-DMPC6 or in DPC micelles,[7] confirms the DMPC bilayer environments of OmpX to be most similar to Salipro[3] (Figure S6) This supports the proper incorporation of the protein into Salipro[3]. At the same time the recovery of peak intensities seems to indicate that the isoprenaline-Nb80-bound ternary complex is conformationally more restricted, comparable to NMR observations for the β2-adrenergic receptor.[30] While these studies show the successful embedding of β1AR in Salipro nanoparticles as a functional receptor, the individual resonance assignments will be presented as the topic of further investigations.
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