Aligned peptoid-based macrodiscs for structural studies of membrane proteins by oriented-sample NMR

Abstract

Development of a robust, uniform, and magnetically orientable lipid mimetic will undoubtedly advance solid-state NMR of macroscopically aligned membrane proteins. Here, we report on a novel lipid membrane mimetic based on peptoid belts. The peptoids, composed of 15 residues, were synthesized by alternating N-(2-phenethyl)glycine with N-(2-carboxyethyl)glycine residues at a 2:1 molar ratio. The chemically synthesized peptoids possess a much lower degree of polydispersity versus styrene-maleic acid polymers, thus yielding uniform discs. Moreover, the peptoid oligomers are more flexible and do not require a specific folding, unlike lipoproteins, in order to wrap around the hydrophobic membrane core. The NMR spectra measured for the membrane-bound form of Pf1 coat protein incorporated in this new lipid mimetics demonstrate a higher order parameter and uniform linewidths compared with the conventional bicelles and peptide-based macrodiscs. Importantly, unlike bicelles, the peptoid-based macrodiscs are detergent free.

Figure 1

(A) Schematic for synthesis of 15-mer peptoid with the chemical formula C₁₂₇H₁₅₀N₁₆O₂₆ and a total molecular weight of 2.3 kDa. (B) Liquid chromatography with tandem mass spectrometry spectrum of the synthesized peptoid demonstrating sample purity at 214 nm.

Figure 2

(A) Dynamic light scattering particle-size distribution by volume for a diluted peptoid macrodisc sample measured at 25°C. Polydispersity index and Z_avg diameter are shown as insets in the top right corner. (B) Transmission electron microscopy images of the peptoid-based macrodiscs loaded with Pf1 coat protein demonstrating the presence of clusters of discs with an average diameter of 40 nm. (C) An enlarged image of an individual disc with the black bar as a scale. To see this figure in color, go online.

Figure 3

(A) ³¹P chemical shift NMR spectrum of the sample of Pf1 coat protein reconstituted in peptoid-belt macrodiscs, at a sample temperature of 37°C and 23:1 lipid:peptoid molar ratio. (B) One-dimensional ¹⁵N NMR spectrum of Pf1 coat protein reconstituted in peptoid-based macrodiscs measured at 37°C.

Figure 4

The effect of temperature on the ³¹P NMR linewidth (A) and resonance position (B) at various lipid:peptoid ratios and presence of the incorporated membrane protein as indicated. To see this figure in color, go online.

Figure 5

(A) Two-dimensional NMR spectra of Pf1 reconstituted in different membrane mimics: the “traditional” q = 3.2 DMPC/DHPC bicelles (green), 14-mer belt peptide-DMPC macrodiscs (red), and 15-mer peptoid-DMPC macrodiscs (blue). (B) Slices through the dipolar dimension for peak G37 from the spectrum of panel A.

Figure 6

(A and B) Chemical shift anisotropy (A) and dipolar (B) line widths measured at half height for each assigned TM residue in the ¹⁵N chemical shift anisotropy and ¹H-¹⁵N dipolar dimensions from two-dimensional NMR spectra: q = 3.2 DMPC/DHPC bicelles (green), belt peptide-DMPC macrodisc (red), and in 15-mer peptoid-DMPC macrodiscs (blue). The connecting lines are shown as a visual guide. More uniform chemical shift anisotropy linewidths are obtained in the case of peptoid-based macrodiscs, indicating a more homogenous rotational motional averaging.