Analytical techniques prevents a more exhaustive comparison, but overall both of

Analytical techniques prevents a more exhaustive comparison, but overall both of them agree. GPI- PrPSc fibrils are about 3? nm wide ([25] and our unpublished results). This constraint means that each PrPSc monomer must be coiled in such a way as to fit approximately 140?45 residues (,G85 232) into this width. To do so, PrPSc monomers must necessarily adopt a multi-layer architecture, as seen in SH3 fibers [26] or the HET-s fungal prion domain [27]. The HET-s prion domain packs 70 residues into two b-strands alternating with turns and loops [27]. Wille et al. have suggested that PrPSc fibrils are composed of four rungs of b-strands, based on their interpretation of X-ray diffraction patterns [28]. In this model, each rung would comprise ,36?7 residues. Positions N152-M153 lie near the middle of the G85-S232 sequence, so it is tempting to speculate that they might be located at an exposed position at the border between rungs. This might explain why the N152-S232 and/or M153-S232 fragment emerges as the most conspicuous PK-resistant fragment after prolonged treatment with PK or partial unfolding with guanidine (Figures 4 and 5). Positions A116-G118 might be the border between the two most aminoterminal rungs (approximately G85-A115 and A119-E151). On the other hand, our results are partially inconsistent with the locationFigure 4. Kinetics of PK digestion of unpurified GPI2 PrPSc. Samples were digested with PK (25 mg/ml) and the reaction stopped after 0, 30, 60, 120, 180, 240, 300 and 360 minutes. Samples were treated with PNGase F and subjected to Tricine-SDS-PAGE the blot was probed with R1 antibody. doi:10.1371/journal.pone.0050111.gStructural Organization of Mammalian PrionsPreparation of Brain Homogenates and Isolation of GPIanchorless PrPScMouse BH, 10 w/v, were 4 IBP manufacturer prepared in PBS, 5 sarkosyl, using a dounce homogenizer (Wheaton Industries Inc, NJ, USA), followed by one pulse of sonication to clarify the homogenate, with an ultrasonic homogenizer probe (Cole Parmer Instrument CO., Chicago IL, USA). GPI2 PrPSc was isolated using the method of Baron et al. [8]. During the purification, total PrPSc was treated with 10 mg/ml of proteinase K. The final GPI2 PrPSc pellet was resuspended in 100 ml of deionised water or in 20 ml of a 6 M guanidine solution (final concentration 1.75 mg/ml). The stock suspension was stored at 4uC. Its purity was assessed by Coomassie stained SDS-PAGE gel and estimated to 18325633 be ,95 pure. The yield of GPI- PrPSc was ,35 mg per brain (BCA protein assay).Figure 5. Western blot of PK-digested series of GPI2 PrPSc samples following partial unfolding by guanidine HCl. After guanidine partial unfolding with 0 M, 0.5 M, 1 M, 2 M, 3 M and 4 M and PK treatment (25 mg/ml), the samples were treated with PNGase F and resolved on Tricine-SDS-PAGE. The WB was probed with the R1 antibody. doi:10.1371/journal.pone.0050111.gRecombinant PrPRecombinant Mouse PrP(23-231) was expressed in E. coli, and purified and refolded Acetovanillone site in-column on an NTA affinity column (GE Healthcare, Uppsala, Sweden), as previously described [29]. Refolded protein was dialyzed against 10 mM sodium phosphate buffer pH 5.8 and then against d.i. water.assigned by Govaerts et al., using threading algorithms, to residues K100-P104 and E145-R163, placed in loops and not rungs [10]. Our data show that the stretches formed by residues K100-P104, N142E151, and Y154-Y161, are PK-resistant, i.e., likely part of a b-strand rung (Figure 2 and Table 1). In summary, our data.Analytical techniques prevents a more exhaustive comparison, but overall both of them agree. GPI- PrPSc fibrils are about 3? nm wide ([25] and our unpublished results). This constraint means that each PrPSc monomer must be coiled in such a way as to fit approximately 140?45 residues (,G85 232) into this width. To do so, PrPSc monomers must necessarily adopt a multi-layer architecture, as seen in SH3 fibers [26] or the HET-s fungal prion domain [27]. The HET-s prion domain packs 70 residues into two b-strands alternating with turns and loops [27]. Wille et al. have suggested that PrPSc fibrils are composed of four rungs of b-strands, based on their interpretation of X-ray diffraction patterns [28]. In this model, each rung would comprise ,36?7 residues. Positions N152-M153 lie near the middle of the G85-S232 sequence, so it is tempting to speculate that they might be located at an exposed position at the border between rungs. This might explain why the N152-S232 and/or M153-S232 fragment emerges as the most conspicuous PK-resistant fragment after prolonged treatment with PK or partial unfolding with guanidine (Figures 4 and 5). Positions A116-G118 might be the border between the two most aminoterminal rungs (approximately G85-A115 and A119-E151). On the other hand, our results are partially inconsistent with the locationFigure 4. Kinetics of PK digestion of unpurified GPI2 PrPSc. Samples were digested with PK (25 mg/ml) and the reaction stopped after 0, 30, 60, 120, 180, 240, 300 and 360 minutes. Samples were treated with PNGase F and subjected to Tricine-SDS-PAGE the blot was probed with R1 antibody. doi:10.1371/journal.pone.0050111.gStructural Organization of Mammalian PrionsPreparation of Brain Homogenates and Isolation of GPIanchorless PrPScMouse BH, 10 w/v, were prepared in PBS, 5 sarkosyl, using a dounce homogenizer (Wheaton Industries Inc, NJ, USA), followed by one pulse of sonication to clarify the homogenate, with an ultrasonic homogenizer probe (Cole Parmer Instrument CO., Chicago IL, USA). GPI2 PrPSc was isolated using the method of Baron et al. [8]. During the purification, total PrPSc was treated with 10 mg/ml of proteinase K. The final GPI2 PrPSc pellet was resuspended in 100 ml of deionised water or in 20 ml of a 6 M guanidine solution (final concentration 1.75 mg/ml). The stock suspension was stored at 4uC. Its purity was assessed by Coomassie stained SDS-PAGE gel and estimated to 18325633 be ,95 pure. The yield of GPI- PrPSc was ,35 mg per brain (BCA protein assay).Figure 5. Western blot of PK-digested series of GPI2 PrPSc samples following partial unfolding by guanidine HCl. After guanidine partial unfolding with 0 M, 0.5 M, 1 M, 2 M, 3 M and 4 M and PK treatment (25 mg/ml), the samples were treated with PNGase F and resolved on Tricine-SDS-PAGE. The WB was probed with the R1 antibody. doi:10.1371/journal.pone.0050111.gRecombinant PrPRecombinant Mouse PrP(23-231) was expressed in E. coli, and purified and refolded in-column on an NTA affinity column (GE Healthcare, Uppsala, Sweden), as previously described [29]. Refolded protein was dialyzed against 10 mM sodium phosphate buffer pH 5.8 and then against d.i. water.assigned by Govaerts et al., using threading algorithms, to residues K100-P104 and E145-R163, placed in loops and not rungs [10]. Our data show that the stretches formed by residues K100-P104, N142E151, and Y154-Y161, are PK-resistant, i.e., likely part of a b-strand rung (Figure 2 and Table 1). In summary, our data.