Fig 3. Dissection of peak I and subsequent analysis.(A) Size exclusion chromatography (SEC) curve of peak I. Imidazole was removed from the peak I sample using dialysis. 45.5 min, 67.2 min, and 79.2 min represented the different retention times of the fractionated species. Buffer C was used here (20 mM Tris, pH 8.0, 1.2 M NaCl). (B) SDS-PAGE for 45.5 min, 67.2 min, and 79.2 min fractions in Figure 3A. The bands in black, blue, and red frames were selected for mass spectrometry. (C) SDD-AGE of peak I SEC fractions. (D) Transmission electron microscopy (TEM) of 45.5 min species from Figure 3A. Lane M in Figure 3B: molecular weight protein marker comprising 100 kDa, 70 kDa, 50 kDa, 40 kDa, and 30 kDa. The buffer used here was composed of 20 mM Tris, pH 8.0, 1.2 M NaCl, and 10 mM DTT. For HiLoad Superdex 200 pg 16/60 size exclusion chromatographic column (1 mL/min), the gel phase distribution coefficient (Kav) of Conalbumin (75 kDa), Aldolose (158 kDa) and Ferritin (440 kDa, Horse Spleen) in Gel Filtration Calibration kit LMW (28-4038-42) were close to 0.38, 0.26 and 0.10, respectively.
Peak I obtained after Ni2+ affinity chromatography could be mainly eluted by SEC at three peaks, with retention times of 45.5 min, 67.2 min, and 79.2 min (Fig 3A). 67.2 min species (gel phase distribution coefficient, Kav, 0.37) is approximate to Conalbumin (75 kDa, 0.36) and the theoretical molecular weight of Sup35 monomer is 76.6 kDa. BN-PAGE (Fig 2C) and SEC curve (Fig 3A) stated that peak I contains Sup35 monomer (67.2 min, Kav, 0.37) and a protein complex (45.5 min, Kav, 0.11), which is an intact complex and not a mixture. This determined 45.5 min does not possess large complexes which all run in the void volume at the same time. Fractionated samples were dissociated into zone X2 bands (red frame in Fig 3B), Sup35 monomer band (black frame in Fig 3B), and X1 band (blue frame in Fig 3B). X1 was identified as ATP synthase β unit (chain D) by mass spectroscopy and zone X2 contained Sup35, chaperone HSP90, Dna K (HSP70), 30 S subunit (Chain C) of E. coli 70 S ribosome, and Omp F (Table S1). SDS-PAGE for 45.5 min (Fig 3B) and further mass spectrum (Table S1) all pointed out that Sup35 protein exists in 45.5 min complex.
Previous studies [27,28] had revealed that Sup35-NM could aggregate through self-assembly into amyloid fibrils, which are relatively high molecular weight polymers featured as smear bands in SDD-AGE. The 45.5 min species had a smear SDD-AGE band, which is fundamentally different from that exhibited by the 67.2 min monomer (Fig 3C). This band pattern elucidated that 45.5 min has aggregated Sup35 (C-aggregates) rather than Sup35 monomers bounded with other protein complexes. Take together, the 45.5 species was assigned as a complex, which was formed by combining Sup35 C-aggregates and zone X2 proteins in E. coli .
The 45.5 min species showed the identical aggregation pattern with Sup35-NM fibril. However, the TEM image of the 45.5 min species had a distinct outlook compared with purified Sup35 fibrils (Fig 3D) [22,28]. HSP90-Dna K-client protein complex was activated during protein misfolding or aggregation, and yeast Ssa1p (HSP90) could inhibit Sup35 monomers to form fibrils [28,29-32]. Here, peak I could be divided in three parts; one was determined to be Sup35 C-aggregate-HSP90-Dna K-30 S ribosomal subunit-Omp F due to the interactions of aggregated Sup35 client with HSP90-Dna K, and the others were ATP synthase β unit (chain D) and Sup35 monomers. The distinctive Sup35 folding states in Sup35 C-aggregate and Sup35 monomer were inherited from the states in E. coli rather than man-made due to the protein purification process. This suggests that HSP90-Dna K could interact with Sup35 aggregates formed in E. coli . Through the influence of Sup35 C-aggregate-HSP90-Dna K-30S ribosomal subunit-Omp F complex on Sup35 purification, we could obtain a portion of Sup35 monomer and ATP synthase using SEC. The Sup35 monomer identified here coincided with some Sup35 monomers dissociated from peak I in BN-PAGE (Fig 2C). All these proofs demonstrate that Sup35 C-aggregate possessed a different in vivo aggregate manner with in vitro -prepared amyloid fibrils.
Fractionated Sup35 monomer could not form an aggregate pattern identical to Sup35 C-aggregate and Sup35-NM fibril. Sup35 monomer could maintain its conformational stability in vitro for a long time and showed an SDD-AGE pattern similar to that observed from the yeast [psi -] monomer state Sup35 (Fig 4A), which indicates that Sup35 monomer was expressed as a conformationally stable species as opposed to Sup35 C-aggregates. They did not possess the ability to form fibrils without being accompanied by the ATP regeneration system [22,33]. The 65.5 min species (Kav, 0.35) was Sup35 monomer and had the same protein state with 67.2 min monomers (Figure 3A). The Kav of 55 min species was 0.22, which was between 0.10 (Aldolose, 158 kDa) and 0.26 (Ferritin, 44 kDa). And, we had previous found that Sup35-NM could form trimer [28]. 55 min species could be low molecular weight aggregates of purified in vitro Sup35 monomers, such as Sup35 trimers (230 kDa). A small amount of the 14-day Sup35 monomer had 55 min species (Fig 4B), which suggested that in vitro Sup35 monomer has a tendency for aggregation, and perhaps the protection system functioned to avoid heterologous prion assembly in E. coli . Sup35 C-aggregates had the 43.5 min retention time after 14-day of storagein vitro (Fig S2), which indicates that the Sup35 C-aggregate-HSP90-Dna K-30 S ribosomal subunit-Omp F complex was not in a state of equilibrium and that this complex blocks Sup35 aggregates from further propagation. This suggests that the Sup35 C-aggregate state fold may be a foreign species in E. coli , which was monitored under the surveillance of the HSP90-Dna K chaperone system.