Macrodiscs Comprising SMALPs for Oriented Sample Solid-State NMR Spectroscopy of Membrane Proteins

Abstract

Macrodiscs, which are magnetically alignable lipid bilayer discs with diameters of >30 nm, were obtained by solubilizing protein-containing liposomes with styrene-maleic acid copolymers. Macrodiscs provide a detergent-free phospholipid bilayer environment for biophysical and functional studies of membrane proteins under physiological conditions. The narrow resonance linewidths observed from membrane proteins in styrene-maleic acid macrodiscs advance structure determination by oriented sample solid-state NMR spectroscopy.

Figure 1

³¹P chemical shift NMR spectra of DMPC bilayers as a function of temperature at a resonance frequency of 283 MHz with ¹H decoupling. (A) Bicelles consist of DMPC and Triton X-100 with q = 5. (B) Macrodiscs consist of DMPC and 14A at a molar ratio of 13.3. (C–E) Macrodiscs consist of DMPC and three different SMA polymers. (C) SMA(1.4:1) are shown with q_d = 7.4. (D) SMA(2:1) are shown with q_d = 27.7. (E) SMA(3:1) are shown with q_d = 49.1. The lipid concentration in all samples is 10% (w/v).

Figure 2

Solid-state NMR spectra of the membrane-bound form of uniformly ¹⁵N-labeled Pf1 coat protein in macrodiscs consisting of DMPC/dimyristoylphosphatidylglycerol (1:1) and SMA(3:1) with q_d = 49.1. The samples are aligned with their bilayer normals (n) perpendicular to the direction of the 21.1 T magnetic field, as illustrated in the cartoon. (A) One-dimensional ¹⁵N chemical shift spectrum was obtained by cross-polarization with a 25 ms acquisition time. (B) Two-dimensional ¹H-¹⁵N dipolar coupling/¹⁵N chemical shift spectrum was obtained using polarization inversion spin exchange at the magic angle with 80 t₁ increments. Both spectra were obtained at 40°C with 45.5 kHz ¹H irradiations and 1 ms cross-polarization mix times.

Figure 3

(A) ¹⁵N chemical shift NMR spectrum of “flipped” Pf1 coat protein in SMA(3:1) macrodiscs was obtained by cross-polarization after addition of 5 mM TmCl₃ to the sample used in Fig. 2. The membrane normal is parallel to the field, as illustrated in the cartoon. (B) ³¹P chemical shift NMR spectrum of DMPC:SMA(3:1) macrodiscs in the presence of 4 mM YbCl₃. (Also shown in Fig. S4)