An Adaptable Phospholipid Membrane Mimetic System for Solution NMR Studies of Membrane Proteins

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.

Figure 1

Biophysical characterization of free SapA, SapA-DDM, and SapA-DMPC nanoparticles. (a) Size-exclusion chromatography. (b) Fluorescence spectroscopy. (c) 1D ¹H NMR spectroscopy. Tryptophan indole signals are indicated by *.

Figure 2

Generation of SapA-DMPC nanoparticles at pH 6. (a) Schematic representation of the SapA-DMPC nanoparticle assembly. (b) SEC of SapA-DMPC with initial mixing SapA:DMPC ratios of 1:15 (blue) and 1:45 (red) at pH 6. DMPC was quantified by molybdate assay (open circle). The proposed SapA arrangement is indicated. (c) Negative-stain EM images of Salipro₃. Scale bar, 50 nm.

Figure 3

2D [¹H,¹⁵N]-SOFAST-TROSY spectrum of non-deuterated ¹⁵N-labeled OmpX in SapA-DMPC nanoparticles.

Figure 4

Overlay of the 2D ¹³C-SOFAST-HMQC spectra of [¹³C_ε-methionine] β₁AR in the apo state (blue), the isoprenaline-bound state (green), and the isoprenaline-Nb80-bound ternary complex (red).