Characterization of nanodisc-forming peptides for membrane protein studies

Abstract

Lipid-bilayer nanodiscs provide a stable, native-like membrane environment for the functional and structural studies of membrane proteins and other membrane-binding molecules. Peptide-based nanodiscs having unique properties are developed for membrane protein studies and other biological applications. While the self-assembly process rendering the formation of peptide-nanodiscs is attractive, it is important to understand the stability and suitability of these nanodisc systems for membrane protein studies. In this study, we investigated the nanodiscs formation by the anti-inflammatory and tumor-suppressing peptide AEM28. AEM28 is a chimeric peptide containing a cationic-rich heparan sulfate proteoglycan- (HSPG)-binding domain from human apolipoprotein E (hapoE) (141-150) followed by the 18A peptide's amino acid sequence. AEM28-based nanodiscs made with different types of lipids were characterized using various biophysical techniques and compared with the nanodiscs formed using 2F or 4F peptides. Variable temperature dynamic light-scattering and ³¹P NMR experiments indicated the fusion and size heterogeneity of nanodiscs at high temperatures. The suitability of AEM28 and Ac-18A-NH_2- (2F-) based nanodiscs for studying membrane proteins is demonstrated by reconstituting and characterizing a drug-metabolizing enzyme, cytochrome-P450 (CYP450), or the redox complex CYP450-CYP450 reductase. AEM28 and 2F were also tested for their efficacies in solubilizing E. coli membranes to understand the possibility of using them for detergent-free membrane protein isolation. Our experimental results suggest that AEM28 nanodiscs are suitable for studying membrane proteins with a net positive charge, whereas 2F-based nanodiscs are compatible with any membrane proteins and their complexes irrespective of their charge. Furthermore, both peptides solubilized E. coli cell membranes, indicating their use in membrane protein isolation and other applications related to membrane solubilization.

Keywords: AEM28; Cell membrane solubilization; NMR; Nanodisc fusion; P450-CPR redox complex; Peptide nanodisc; Peptide:lipid interaction.

Figure 1.. Self-assembly of amphipathic peptides and lipids to form nanodiscs.

(A) Amino acid sequences of AEM28, 2F, and 4F peptides investigated in this study. The 100 % conserved residues in these peptides are indicated with *. (B) Solubilization of DMPC liposomes using AEM28. The sample was prepared using 10 mg lipids and 10 mg peptide (in a 1:1 (w/w) ratio). The photographs were taken at room temperature. (C) SEC chromatogram of the AEM28:DMPC self-assemblies. (D) Insoluble aggregates of AEM28-DMPC-DMPG (1:0.5:0.5 w/w ratio). (E & F) 2F:DMPC (E) and 4F:DMPC (F) self-assemblies. The samples were prepared using a 1:1 (w/w) ratio of lipids to peptides.

Figure 2.. ¹H NMR and DLS characterization of peptide-based nanodiscs.

(A) ¹H NMR spectrum of the SEC-purified AEM28:DMPC complex. The region between 5.8 and 11.5 ppm is expanded to show the amide/aromatic peaks from AEM28 (inset). The DMPC peaks are labelled with assignments. The high-intensity peak at 3.7 ppm was from Tris buffer. The peaks from AEM28 are labelled. (B, C & D) DLS profiles of the SEC-purified AEM28:DMPC (B), 2F:DMPC (C), and 4F:DMPC (D) self-assemblies. All the samples were prepared with 1:1 w/w peptide:lipid ratios.

Figure 3.. Peptide-lipid interactions by NMR.

(A-C) ¹H NMR spectra of AEM28 (bottom) and 1:0.25 w/w AEM28:DMPC nanodiscs. For clarity, amide and aromatic regions of ¹H NMR spectra are shown; the peptide peaks in these regions are not overlapping with lipid peaks like those in the aliphatic region. The NMR chemical shift changes for 1:0.25 w/w AEM28:DMPC nanodiscs (top) compared to the free AEM28 peptide (bottom) (amide-H^N and aromatic protons) are indicated with dashed vertical lines. The large downfield chemical shift change (0.23 ppm) observed for Trp12 aromatic side chain H^N is indicated with an arrow. ‘*’ indicates the peak-broadening due to conformational heterogeneity of AEM28 in the absence of lipids. (D-G) Selected regions of 2D NOESY spectra of 1:0.25 w/w AEM28:DMPC nanodiscs (black) and AEM28 (red). The internuclear DMPC-AEM28 NOE cross-peaks (Trp12, Phe28/DMPC-CH₂) and lipid-induced chemical shift changes (Phe28, Val20, Ala21, Ala27) are labelled with partial peak-assignments. The lipid-induced disappearance of cross-peaks in the Lys/Arg side chain region (E) is boxed.

Figure 4.. Fusion of peptide-based nanodiscs.

(A) A bar graph depicting the hydrodynamic radii of AEM28:DMPC self-assemblies calculated from variable-temperature DLS profiles. The data plotted were collected at nine different temperatures ranging from 20 to 60 °C with 5 °C intervals. The sample was pre-heated and equilibrated for 5 minutes before recording the data at each temperature. (B) DSC analysis of 1:1 w/w AEM28:DMPC assemblies (red) and DMPC liposomes (black); the heat capacity (C_p) values are normalized. The physical phase of DMPC lipids below (ripple phase) and above (liquid crystalline phase) the gel-to-lamellar phase transition (T_m) is schematically depicted.

Figure 5.. Secondary structure of peptides forming the belt of nanodiscs.

Conformational analysis of 1:1 w/w 2F:DMPC nanodiscs (A), AEM28 (B), and 1:3 w/w AEM28:DMPC (C) . The samples were prepared in 10 mM Tris buffer (pH 7.4), and the spectra were recorded at 30 °C.

Figure 6.. ³¹P NMR of peptide-based nanodiscs.

Variable temperature ³¹P NMR spectra of (A) 1:1 w/w AEM28:DMPC (3 mg/mL), (B, C) 1:3 w/w AEM28:DMPC (25 mg/mL AEM28 and 75 mg/mL DMPC) and (D, E) 1:3 w/w AEM28:DMPC (50 mg/mL AEM28 and 150 mg/mL DMPC) nanodiscs. The spectra were recorded using a 400 MHz Bruker solid-state NMR spectrometer equipped with a 5 mm triple-resonance HXY MAS NMR probe. The samples were prepared in 10 mM Tris buffer (pH 7.4), and the spectra were recorded at the indicated sample temperatures. The isotropic peaks are indicated with the down arrows at the top (0 ppm). Above ~300 K, the peak shifted to the high field ((−6.3 ppm in D) or (−7 ppm in E)), likely due to the magnetic-alignment of nanodiscs (indicated by the dashed vertical lines). In addition, the broad powder pattern observed at high temperatures (>~305 K) indicates the existence of a combination of aligned (at least partially) and unaligned lipid aggregates (or vesicles), indicating disorder in the orientations due to random collisions and fusion of nanodiscs enabled by thermal energy.

Figure 7.. Activity of CYP450 and CYP450-CPR redox complex reconstituted in peptide-based nanodiscs.

(A) Reduction of CYP450 2B4 reconstituted in 1:1 w/w AEM28:DMPC nanodiscs characterized by CO-assay shown by UV/vis absorption spectra. The UV/vis spectra of oxidized (Fe³⁺) CYP450 2B4 (black), reduced (Fe²⁺) CYP450 2B4 (cyan), and the CO-bound CYP450 2B4 (Fe²⁺–CO) (red). The shift in Soret peak from 417 nm to 451 nm in the presence of CO and sodium dithionite indicates the functionally stable form of CYP450 2B4 in 1:1 w/w AEM28:DMPC nanodiscs. The low-intensity peak that appeared at ~423 nm may be due to an incomplete reduction of the protein or from a small portion of an inactive form of the protein present in the sample. The spectra were measured at room temperature in a 10 mM Tris (pH 7.4) buffer. (B, C) Photographs showing turbid/precipitated solutions that were formed when 1:1 w/w AEM28:DMPC nanodiscs with CYP450 were mixed with anionic CPR (B) and cytochrome b5 (C). (D) The soret-band region in the absorption spectra of CO-bound CYP450 2B4 shows a time-dependent decrease in the peak intensity at 417 nm and the appearance of a new peak at 451 nm (indicated with dotted arrows). (E) Kinetic traces (@451.25 nm) from the reduced (Fe²⁺) CO-bound CYP450 2B4 in the presence of CPR and benzphetamine.

Figure 8.. Solubilization of membrane by nanodisc-forming peptides.

SDS-PAGE analysis of E. coli membrane solubilization by AEM28 and 2F peptides. The intense band (circled) is likely from AEM28 aggregates formed by its interaction with SDS. The labels I, S, and M denote insoluble membrane fraction, peptide-solubilized membrane fraction, and protein marker, respectively.