Fig. 6

Evaluation of the effect of glass bead size, amount, and surface on spontaneous misfolding efficiency by PMSA. A The effect of bead size on spontaneous misfolding was first evaluated using glass beads of 0.1 mm and 1 mm diameters, with the same weight (approximately 30 mg of beads per tube) for both types. Four independent experiments were set with each bead type, and three serial 24h PMSA rounds (R1 to R3) were performed. The product from the previous round was used to seed subsequent passages at a 1:10 dilution. Spontaneous rec-PrP^res formation was evaluated through proteinase K digestion and electrophoresis, followed by total protein staining. rec-PrP^res could be detected from the first round when 0.1 mm beads were used, whereas 1 mm diameter beads required at least two serial rounds to induce detectable levels of misfolding. This suggests that the amount of glass surface area could be a relevant parameter for efficient misfolding occurrence. B The effect of bead amount on spontaneous misfolding was examined by using exact numbers of 1 mm diameter glass beads in each reaction tube. PMSA substrate was supplemented with 0, 1, 2, 4, 8, 16, 32 and 64 beads, and each tube was subjected to three serial 24 h PMSA rounds (R1 to R3), with a 1:10 dilution between rounds. Results obtained by proteinase K digestion, electrophoresis, and total protein staining indicate that a minimum of 8 beads is required to give rise to rec-PrP^res in the first PMSA round, highlighting the necessity of a sufficient number of beads for efficient spontaneous misfolding in PMSA. C To definitively demonstrate the critical parameter for highly efficient spontaneous misfolding of rec-PrP, we conducted experiments using glass beads of two different sizes: 0.2 and 1 mm diameter. These beads were used in quantities that achieved equivalent surfaces areas of 10π, 25π, 100π, 250π, 1000π, and 2500π mm². To ensure robustness, four replicate tubes were used for each surface and bead size. All tubes underwent a single 24 h PMSA round. Subsequently, the formation of rec-PrP^res in each tube was determined through proteinase K digestion, electrophoresis and total protein staining. The resulting data was plotted, representing the percentage of rec-PrP^res positive replicates for each condition. It is worth noting that the inclusion of the required amount of 1 mm diameter beads to achieve surfaces areas of 1000π and 2500π mm² hindered the free movement of beads inside the tubes. Consequently, the negative results obtained from these conditions were considered inconclusive and were omitted from the plot denoted by an asterisk (*). Our analysis revealed an optimal range of glass surface area between 100π mm² and 250π mm², irrespective of the bead size. This range could potentially extend up to 1000π mm². Surfaces below and above this range showed reduced efficiency in rec-PrP misfolding indicating the existence of a minimum glass surface area that negatively affects spontaneous rec-PrP misfolding efficiency. This reduction may be attributed to the depletion of soluble rec-PrP from the substrate