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. 2020 Jan 21;118(2):403-414.
doi: 10.1016/j.bpj.2019.11.3381. Epub 2019 Nov 28.

Folding of the β-Barrel Membrane Protein OmpA into Nanodiscs

DeeAnn K Asamoto  1 , Guipeun Kang  1 , Judy E Kim  2
Affiliations

Folding of the β-Barrel Membrane Protein OmpA into Nanodiscs

DeeAnn K Asamoto et al. Biophys J. .

Abstract

Nanodiscs (NDs) are an excellent alternative to small unilamellar vesicles (SUVs) for studies of membrane protein structure, but it has not yet been shown that membrane proteins are able to spontaneously fold and insert into a solution of freely diffusing NDs. In this article, we present SDS-PAGE differential mobility studies combined with fluorescence, circular dichroism, and ultraviolet resonance Raman spectroscopy to confirm the spontaneous folding of outer membrane protein A (OmpA) into preformed NDs. Folded OmpA in NDs was incubated with Arg-C protease, resulting in the digestion of OmpA to membrane-protected fragments with an apparent molecular mass of ∼26 kDa (major component) and ∼24 kDa (minor component). The OmpA folding yields were greater than 88% in both NDs and SUVs. An OmpA adsorbed intermediate on NDs could be isolated at low temperature and induced to fold via an increase in temperature, analogous to the temperature-jump experiments on SUVs. The circular dichroism spectra of OmpA in NDs and SUVs were similar and indicated β-barrel secondary structure. Further evidence of OmpA folding into NDs was provided by ultraviolet resonance Raman spectroscopy, which revealed the intense 785 cm-1 structural marker for folded OmpA in NDs. The primary difference between folding in NDs and SUVs was the kinetics; the rate of folding was two- to threefold slower in NDs compared to in SUVs, and this decreased rate can tentatively be attributed to the properties of NDs. These data indicate that NDs may be an excellent alternative to SUVs for folding experiments and offer benefits of optical clarity, sample homogeneity, control of ND:protein ratios, and greater stability.

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Figures

Figure 1
Figure 1
The top shows the membrane topology of OmpA. Arginine residues are indicated in bold and are underlined. The bottom shows the primary sequence of 14A, the belt peptide.
Figure 2
Figure 2
SDS-PAGE result of OmpA digestion. Gels show digestion of OmpA in NDs (A) and in SUVs (B). The lipid:protein ratio was 300:1 and the residual urea concentration was 0.3 M before digestion. Samples during folding and digestion were incubated at 37°C. For both gels: lane 1: folded in NDs (A) or in SUVs (B); lane 2: folded in NDs (A) or SUVs (B) and digested with Arg-C for 3 h; lane 3: folded in NDs (A) or SUVs (B) and digested with Arg-C for 24 h; lane 4: unfolded (unf) OmpA in 3.6 M urea; and lane 5: unf OmpA in 3.6 M urea and digested with Arg-C for 4 h. The bands for folded, unf, and the ∼26- and ∼24-kDa transmembrane fragments (TM26 and TM24) are indicated.
Figure 3
Figure 3
Arg-C digestion of wild-type OmpA adsorbed on NDs (top) and SUVs (bottom) at 16°C. In each image, the left lane contains protein, and the right lane is the molecular weight ladder. Gels are of OmpA adsorbed on (A) NDs, no digestion; (B) NDs, 4-h digestion; (C) NDs, 24-h digestion; (D) SUVs, no digestion; (E) SUVs, 4-h digestion; and (F) SUVs, 24-h digestion. The residual urea concentration in the samples before digestion was 0.2 M.
Figure 4
Figure 4
Wild-type OmpA folding reaction monitored by SDS-PAGE at 33°C for 4 h. Data were collected at 4, 8, 16, 30, 46, 60, 120, 180, and 240 min after initiation of the folding reaction. The top shows SDS-PAGE gels of OmpA folding in the presence of (A) NDs and (B) SUVs. The bottom shows fraction folded based on gel band densities for OmpA in NDs (triangles) and SUVs (solid circles). The lipid:protein ratio in SUVs and NDs was 400:1 and the residual urea concentration was 0.2 M. The curves and resulting folding times are based on single exponential fits to the data, including a value of 0 at time 0 min.
Figure 5
Figure 5
Wild-type OmpA folding reaction monitored by fluorescence spectroscopy at 33°C for 4 h. Spectra were acquired 1, 4, 8, 16, 30, 46, 60, 120, 180, and 240 min after initiation of the folding reaction in DMPC NDs (A) or SUVs (B). The lipid:protein ratio was 400:1 and the residual urea concentration was 0.2 M for both samples. The steady-state spectra of OmpA folded in NDs and SUVs and unfolded in 8 M urea are shown in (C). The fraction folded during folding reactions in SUVs (solid circles) and NDs (triangles) are shown in (D), with single exponential fits (including a value of 0 at time 0 min).
Figure 6
Figure 6
UVRR difference spectra. Signal from the buffer, urea, SUVs, and NDs as well as general scattering have been subtracted for OmpA W129 mutant in (A) 0.8 M urea, (B) adsorbed on DPPC SUVs, (C) folded in detergent (OG), (D) folded in DMPC SUVs, and (E) folded in DMPC NDs. The spectrum of ND-only in which signal from buffer is subtracted is shown as (F). All spectra were collected at room temperature and for 10 min, with the exception of the spectrum of OmpA in NDs (E), which was collected for 5 min. The region near 1000 cm−1 (indicated with ∗) has strong urea signal in each sample except (F), resulting in a subtraction artifact.
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