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. 2024 Feb 13;12:RP92307. doi: 10.7554/eLife.92307

Figure 3. HiSiaQM self-association in lauryl maltose neopentyl glycol (L-MNG) and amphipol.

(a) Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis of HiSiaQM in L-MNG (left panel). Two well-resolved species exist at 7.3S (diffusion coefficient, D = 4.8 × 10–7 cm2/s) and 9.9S (D = 4.2 × 10–7 cm2/s), with the larger peak constituting 85% of the signal. The species at 7.3S (peak 1, blue shading) is most consistent with HiSiaQM as a monomer with ~98 molecules of L-MNG bound (middle panel; green = measured mass, black = theoretical mass), calculated from the experimental sedimentation and diffusion coefficients. These calculations suggest that peak 1 existing as a dimer is unlikely, as the dimeric protein would only have ~14 molecules of L-MNG bound. Additionally, the calculated f/f0 of a monomer for peak 1 is 1.2, consistent with a protein in a detergent micelle. The species at 9.9S (peak 2, pink shading) is most consistent with HiSiaQM as a dimer with ~116 molecules of L-MNG bound (calculated) (right panel; purple = measured mass, black = theoretical mass); peak 2 existing as a monomer is not possible, as the protein clearly has a smaller species in peak 1 and cannot be divided further than a monomer, and a trimer is also unlikely as the trimeric protein would only have ~32 molecules of L-MNG bound (calculated). Additionally, the calculated f/f0 of a dimer for peak 2 is also 1.2, again consistent with a protein in a detergent micelle. These calculations do not account for bound lipid molecules. (b) Left panel: SV-AUC analysis of amphipol-solubilised HiSiaQM (initially purified in L-MNG) shows two distinct species present at 5.9S and 8.3S. These are monomeric and dimeric species as L-MNG-solubilised protein exists as these oligomeric states at 7.3S and 9.9S as in (a). Right panel: representative size-exclusion chromatogram of amphipol-solubilised HiSiaQM favouring the dimeric state. The main peak at ~10.8 mL contains dimeric HiSiaQM and the shoulder at ~11.8 mL contains monomeric HiSiaQM. The sample used for structure determination is shaded turquoise.

Figure 3.

Figure 3—figure supplement 1. HiSiaQM self-association in dodecyl-β-d-maltoside (DDM).

Figure 3—figure supplement 1.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis of HiSiaQM in DDM (left panel). Two well-resolved species exist at 7.6S (diffusion coefficient, D = 5.0 × 10–7 cm2/s) and 10.3S (D = 5.0 × 10–7 cm2/s), with the larger peak constituting 62% of the signal. Due to the presence of protein aggregation in DDM (Figure 2—figure supplement 1a, green trace), SV-AUC for this sample was performed at 4°C (instead of 20°C for lauryl maltose neopentyl glycol [L-MNG]) to prevent HiSiaQM from forming larger order species, although even at this temperature larger species are still evident at 12–18S and extend to ~40S (not shown). This observed heterogeneity was a significant reason for our use of L-MNG in all other experiments. The species at 7.6S (peak 1, blue shading) is most consistent with HiSiaQM as a monomer with ~216 molecules of DDM bound (middle panel; green = measured mass, black = theoretical mass), calculated from the experimental sedimentation and diffusion coefficients. These calculations suggest that peak 1 existing as a dimer is unlikely, as the dimeric protein would only have ~31 molecules of DDM bound. Additionally, the calculated f/f0 of a monomer for peak 1 is 1.1, consistent with a protein in a detergent micelle. The species at 10.3S (peak 2, pink shading) is most consistent with HiSiaQM as a dimer with ~171 molecules of DDM bound (calculated) (right panel; purple = measured mass, black = theoretical mass); peak 2 existing as a monomer is not possible as the protein clearly has a smaller species in peak 1 and cannot be divided further than a monomer, and a trimer is also unlikely as the experimental data suggests that no DDM would be bound (calculated). Additionally, the calculated f/f0 of a dimer for peak 2 is 1.0, roughly consistent with a protein in a detergent micelle. These calculations do not account for bound lipid molecules.
Figure 3—figure supplement 2. HiSiaQM self-association in lauryl maltose neopentyl glycol (L-MNG) (interference analysis).

Figure 3—figure supplement 2.

Sedimentation of HiSiaQM in L-MNG (left panel) with absorbance data (pink) and interference data (green). Two well-resolved species exist at 6.7S and 9.4S, with the smaller species constituting greater than 80% of the signal. The oligomeric state of the two species was verified using the membrane protein calculations function of GUSSI (Brautigam, 2015), as described previously (Ebel, 2011; le Maire et al., 2008; Le Roy et al., 2015; Salvay et al., 2007). Free L-MNG micelles are seen only with interference data at ~3.2S. The peak at 6.7S is most consistent with a HiSiaQM monomer (middle panel). After determining the amount of L-MNG bound to the protein with the interference data, frictional ratios (f/f0) can be calculated to test the hypothesised oligomeric state. The calculated f/f0 for a monomer for the major species (red shading) is 1.2 (1σ error = 1.13–1.24), consistent with a single HiSiaQM in a detergent micelle. A dimer is unlikely since the calculated f/f0 for a dimer for the major species is 1.9 (1σ error = 1.79–1.96) (not shown). On the right panel, the calculated f/f0 for a dimer for the minor species (blue shading) is 1.3 (1σ error = 1.23–1.35), again consistent with dimeric HiSiaQM in a detergent micelle. A monomer is unlikely, since the calculated f/f0 for a monomer for the 9.4S species is 0.8 (1σ error = 0.78–0.85) (not shown). With the detergent bound to the sedimenting protein now known alongside the mass of the detergent monomers, it can be estimated that approximately 90 (monomer) and 150 (dimer) molecules of L-MNG are bound to the protein.
Figure 3—figure supplement 3. HiSiaQM self-association in dodecyl-β-d-maltoside (DDM) (interference analysis).

Figure 3—figure supplement 3.

Sedimentation of HiSiaQM in DDM (left panel) with absorbance data (pink) and interference data (green). Two main species exist at ~4.5S and ~6.2S, with the smaller species constituting greater than 60% of the signal. The oligomeric state of the two species was verified using the membrane protein calculations function of GUSSI (Brautigam, 2015), as described previously (Ebel, 2011; le Maire et al., 2008; Le Roy et al., 2015; Salvay et al., 2007). Free DDM micelles are seen only with interference data at ~2.1S. Due to the presence of protein aggregation in DDM (Figure 2—figure supplement 1a, green trace), sedimentation velocity analytical ultracentrifugation (SV-AUC) for this sample was performed at 4°C (instead of 20°C for the sample in lauryl maltose neopentyl glycol [L-MNG]) to prevent the protein from forming larger order species, although even at this temperature some larger species are still evident at ~8–12S. These sedimentation coefficients are not corrected for the lower temperature so appear smaller than all other values reported for HiSiaQM. The species at 4.5S (red shading) is consistent with a monomer in DDM, f/f0 = 1.1 (1σ error = 1.06–1.15) (middle panel). In contrast, a dimer species is unlikely given the unrealistic f/f0 = 1.8 (1σ error = 1.68–1.82) (not shown). The larger species at 6.5S (blue shading) is consistent with a dimer in DDM, f/f0 = 1.2 (1σ error = 1.19–1.3) (right panel), whereas the monomer provides an unrealistic f/f0 = 0.8 (1σ error = 0.75–0.82) (not shown). With the detergent bound to the sedimenting protein now known alongside the mass of the detergent monomers, it can be estimated that approximately 220 (monomer) and 380 (dimer) molecules of DDM are bound to the protein.
Figure 3—figure supplement 4. Solubilisation of HiSiaQM in nanodiscs.

Figure 3—figure supplement 4.

(a) The size-exclusion chromatogram following nanodisc reconstitution with a 1:4:80 ratio of HiSiaQM:MSP:lipid identifies the presence of multiple species. Three fractions across the elution profile were analysed with sedimentation velocity analytical ultracentrifugation (SV-AUC) (shown by the coloured bars). (b) SV-AUC analysis of nanodisc solubilised HiSiaQM (pink, brown, and green) and empty nanodiscs (black) shows that later eluted fractions gave a main species consistent with empty nanodiscs (pink, 4.0S), middle eluted fractions gave a main species consistent with a monomer incorporated (brown, 6.8S) and earlier eluted fractions gave a larger 9.0S main species (green). The larger species at 9.0S is most likely a dimer of HiSiaQM in a nanodisc as it has been shown to exist as a dimer in both detergent and amphipol. The dimer can just fit by physical constraints as the MSP forms ~11 nm nanodiscs and the size of the HiSiaQM dimer is ~10 nm at the widest point. While physically possible, the number of reconstituted lipids would be low.