Membrane proteins in magnetically aligned phospholipid polymer discs for solid-state NMR spectroscopy

Abstract

Well-hydrated phospholipid bilayers provide a near-native environment for membrane proteins. They enable the preparation of chemically-defined samples suitable for NMR and other spectroscopic experiments that reveal the structure, dynamics, and functional interactions of the proteins at atomic resolution. The synthetic polymer styrene maleic acid (SMA) can be used to prepare detergent-free samples that form macrodiscs with diameters greater than 30 nm at room temperature, and spontaneously align in the magnetic field of an NMR spectrometer at temperatures above 35 °C. Here we show that magnetically aligned macrodiscs are particularly well suited for solid-state NMR experiments of membrane proteins because the SMA-lipid assembly both immobilizes the embedded protein and provides uniaxial order for oriented sample (OS) solid-state NMR studies. We show that aligned macrodiscs incorporating four different membrane proteins with a wide range of sizes and topological complexity yield high-resolution OS solid-state NMR spectra. The work is dedicated to Michelle Auger who made key contributions to the field of membrane and membrane protein biophysics.

Keywords: Macrodisc; Membrane proteins; SMA; SMALP; Solid-state NMR.

Figure 1.. Preparation of membrane proteins in magnetically aligned macrodiscs and size characterization of SMA macrodiscs.

(A) Steps 2–4 are interdependent (double arrows) and require optimization. (B, C) Dynamic light scattering profiles of macrodisc size distribution versus scattering number for (B) DMPC liposomes (83.2 ± 24.6 nm) or (C) DMPC/SMA(3:1) (10/3, w/w) SMA macrodiscs (31.4 ± 8.8 nm). Different colors represent three independent measurements made for each sample. These protein-free samples are representative of protein-loaded samples. Inset images illustrate the transition to a translucent preparation that is a hallmark of macrodisc formation. DLS experiments were performed at 25°C. The lipid concentration was 1% (w/v).

Figure 2.. Solid-state NMR spectra of four membrane proteins in magnetically aligned SMA macrodiscs.

(A-D) Representations of the protein structures after 100 ps of dynamics with eefxPot, where the starting structures were determined by solid-state NMR (A, B, C) or solution NMR (D) in lipid bilayers: Pf1 major coat protein (PDB: 2KSJ), MerFt (PDB: 2LJ2), Ail (PDB: 5VJ8), and CXCR1 (PDB: 2LNL). The gray horizontal lines depict the membrane boundaries. The structure of pF1 was calculated with the assigned data from the SLF spectrum (M) in SMA macrodiscs. (E-H) ³¹P NMR spectra. (I-L) ¹⁵N NMR spectra. (M-P) Two-dimensional ¹H/¹⁵N SLF spectra. The spectrum of Pf1 was assigned by comparison with the spectra from bicelles. (Q-U) Back-calculated ¹H/¹⁵N SLF spectra derived from the membrane-embedded protein structures. Peaks from sidechains and N- and C-termini are not shown. For Pf1, the experimental SLF spectrum was assigned by direct comparison to the previously assigned SLF data from bicelles [45], and the assigned SMA data were used to restrain the dynamics for back-calculation. For MerFt and CXCR1, the dynamics were restrained by directly applying the experimental values of ¹⁵N chemical shift and ¹H/¹⁵N dipolar coupling measured previously in bicelles or liposomes [47, 52]. For Ail, the dynamics were not restrained and the protein was allowed to adopt the preferred orientation dictated solely by the membrane potential of eefxPot.