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A wide range of PSI purification protocols from plant material are available in the literature and have produced samples suitable for high-resolution structural determination (e.g. Amunts et al., 2007; Mazor et al., 2017; Qin et al., 2011, 2015). Here the method described by Mazor et al., 2017 for isolation of PSI from pea leaves was adapted for spinach. In brief, unstacked thylakoid membranes were isolated from spinach leaves and treated with αDDM for 5 minutes, to selectively deplete PSII, ATP synthase and cytb6f. After centrifugation, the pellet was taken and frozen at -80°C. Following

thawing, the membranes were solubilised with 1.5% (w/v) βDDM and centrifuged again to pellet insoluble material. The supernatant was applied to a DEAE anion exchange column, and an increasing salt concentration gradient was applied. Dark green fractions with PSI absorbance spectra were pooled and precipitated using 10% PEG6000, followed by centrifugation. The pellet was then resuspended and applied to a 10-35% sucrose gradient (Figure 5.2A). The dark green band (indicted by an arrow) was extracted, and a DEAE anion column was used to concentrate the sample and remove sucrose prior to being applied to another 10-35% sucrose gradient (figure 5.2B). The single band from this was extracted, and frozen until further use.

130 Figure 5.2 Mazor PSI purification method

Purification process for the Mazor et al., 2017 method of purification of untagged PSI. (A) Initial sucrose gradient performed on the PEG6000 precipitated material. The dark band highlighted was extracted, had sucrose removed via DEAE column, and was run on a second sucrose gradient, producing the single band seen in (B). (C) SDS-PAGE of the stages of the purification to observe any loss or changes in material quality. Lanes: 1) βDDM solubilised material supernatant, 2) DEAE column loading flow through, 3) DEAE column PSI fractions pooled, 4) End purified product, showing only PsaA/B. (D) SDS-PAGE of the sample in lane 4 in (C) stained with Sypro-Ruby protein stain for higher sensitivity.

Figure 5.2C shows the SDS-PAGE profile of each step during the purification process. It is clear that a significant amount of PSI is not binding to the DEAE column under these conditions since it is present in the flow through prior to application of the gradient (lane 2). Indeed, compared to the sample loaded onto the column (lane 1) the eluted fractions (lane 3) shows little further enrichment in PSI. Moreover, following PEG6000-induced precipitation, only the PSI core subunits (PsaA/B) are obvious on the gel (lane 4, and D) suggesting that the minor subunits are lost during the purification.

Following these results, the purification process was simplified by removing the PEG 6000 precipitation. Instead, following the selective enrichment by αDDM and the subsequent solubilisation of the enriched sample by βDDM, the supernatant was concentrated and applied to a single 10-35% sucrose gradient. The resultant sucrose gradient can be seen in figure 5.3A, with the extracted bands

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indicated as middle (red) and bottom (black). Following this, absorbance spectra were taken of the samples, shown in figure 5.3B. Both the middle and bottom fractions had noticeably PSI-like absorption spectra, with chlorophyll Qy peaks around 680nm (Ruban, 2013). The middle band had a

slight blue shift from 680nm, which can be indicative of contamination with PSII, which has a spectral peak of 675nm (Ruban, 2013). SDS-PAGE (figure 5.3C) was performed on both samples, showing that the middle band contained a 37kDa size protein (labelled as unknown contaminant) not known to be a part of PSI, and was proposed to be CP43/47 from PSII, which would have blue-shifted the absorption spectrum as previously mentioned. The bottom fraction appeared to be cleaner, and as such was taken forward in the purification.

132 Figure 5.3 Simplified PSI purification method

(A) Sucrose gradient of βDDM solubilised material, producing 3 bands. As the top one was already known to contain LHCII (Crepin et al., 2016), the bottom two bands were taken. (B) Absorbance spectra for the bands extracted in (A). Dashed lines shown are 675nm (green) and 680nm (blue), indicating of PSII and PSI absorbance spectra peaks respectively (Ruban, 2013). Inlay shows magnified region for peaks. (C,D) SDS PAGE of samples throughout the purification process. (C) 2 bands from (A/B). (D) SDS- PAGE of the purification process. Bands: 1) Thylakoid membranes, 2) αDDM membrane pellet 3)

133 Bottom band from sucrose gradient in (A), 4) DEAE column flow through following sample loading, 5) Concentrated PSI fractions from DEAE column.

The bottom band was applied to the DEAE column run in the previous iteration of the purification. Observing the protein complement before (figure 5.3D, lane 3) and after (lane 5) the column did not see a noticeable increase in purity of PSI. In addition, some PSI material is seen to be lost in the flow through loading the DEAE column (lane 4). For an additional measure, the bottom band from the sucrose gradient and the end PSI sample following the DEAE column were also tested via 77k fluorescence, seen in figure 5.4.

Figure 5.4 77K Fluorescence for PSI purification

77K fluorescence of (A) the bottom sucrose gradient band seen in figure 5.3A and (B) the final purification product of fractions pooled from the DEAE column. The peak at 740nm is indicative of PSI, whilst the peak at 675nm is indicative of PSII. No significant change in purity was observed here from the DEAE column.

The emission peak at ca. 740nm is indicative of PSI complex, whereas the 680 nm emission is from LHCII/LHCI (Lamb et al., 2015). This showed the DEAE column was not providing any further enrichment of PSI and indeed the SDS-PAGE in figure 5.3D shows that PSI was eluting at the loading stage of the column. As such the DEAE column was excluded from the purification process, and the

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protocol would simply comprise of the solubilisation steps, followed by the sucrose gradient (materials and methods section 2.4.3).

Whilst the method would only require a single sucrose gradient when done on small samples, the scaling up of the purification to attain more material led to a decreased resolution on the sucrose gradient. As such, when using the larger SW32 rotor, 2 sucrose gradients were performed, with the first one (10-50%) producing 2 bands, comprised of the ‘top’ and ‘middle + bottom’ bands seen in figure 5.3. The bottom of the two bands was taken, and following desalting to remove sucrose, a second gradient (25-40%) also yielded two bands as seen in figure 5.5A. Once again performing 77K fluorescence on these two bands showed that the upper band presented a LHCII like emission spectrum, whilst the bottom band possessed the characteristic emission spectrum for PSI (figure 5.5B/C). Following this the lower band of the gradient was frozen at -80 °C until use, retaining the sucrose as a cryoprotectant. For further details on the purification process, see materials and methods section 2.4.4. In addition, an SDS PAGE was run on this sample, shown in figure 5.5D. The bands labelled clearly match those found previously from the purification of PSI-LHCI complex (Qin et al., 2011).

Figure 5.5 Scaled up purification of PSI

(A) Second sucrose gradient from PSI purification, with the upper and lower bands indicated by red and black respectively. (B/C) 77K fluorescence of the bands seen in (A) (D) SDS – PAGE showing the end purification product following the PSI protocol, with bands labelled matching those seen in (Qin et al.,

135 2011) showing the SDS-PAGE visible components of the PSI – LHCI complex. (E) Western blot bands for PsaF and PsaN subunits on PSI.

The intactness of the Pc binding site on the purified PSI complex was confirmed by immunoblotting the sample for the presence of the labile PsaF and PsaN subunits (figure 5.5E). The purified PSI complex was quantified via a Lowry protein assay for use in further experiments. For details on the assay, see materials and methods section 2.3.8.