Calcium Dependent Prion Protein Toxic Conformation Figure 4. Correlation between toxicity and membrane conduc- tance of hPrP90–231 peptide. All the experimental conditions data reported can be compared with the PrP27–30 protein data (1) representing the native pathological prion protein. Data are plotted as linear regression and statistical evaluation was performed by ANOVA. doi:10.1371/journal.pone.0038314.g004 aggregation process. These results are in line with our previous reports showing that long thermal denaturation treatments favors hPrP90–231 aggregation on preamyloid fibrils [48] that cannot be internalized by the cells and are not cytotoxic [48,49]. Thus, we can hypothesize that these macroaggregates/prefibrillar structures may less efficiently interact also with the artificial lipid bilayer to induce a reduced ionic conductance. Effects of Ca++ Binding of hPrP90–231 Structure To directly monitor the effects of Ca++ on hPrP90–231 structure, we analyzed several parameters that previously we demonstrated to be determinant for the gain of toxicity of hPrP90231. By CD analysis, we demonstrated that hPrP90–231, which in its native conformation is mainly a-helix structured (Fig. 6A), in the presence of increasing [Ca++], loses its structuration. A similar structural change occurred after thermal denaturation at 53uC [7]. Higher [Ca++] caused a reduction of the spectroscopic signal likely due to the aggregation of the peptide. By means of ThT binding assay, as index of b-sheet content, we also show that incubation of hPrP90–231 with 10 mM Ca++ induced an increase in the fluorescence emission (C-max) slightly higher than that induced by thermal denaturation (Fig. 7), suggesting the likely occurrence of increasing in the b-sheet content as a consequence of incubation in high [Ca++] as demonstrated after thermal denaturation. In fact, no additivity on ThT binding was observed combining the two treatments on hPrP90–231 (Fig. 7). hPrP90–231 N-terminus structural unfolding in the presence of high [Ca++] was also monitored by measuring the intrinsic fluorescence of the unique tryptophanylic residue located in the position 99 of the hPrP90–231 sequence. Incubation of the peptide with 1.8, 5 and 10 mM Ca++ caused a concentration-dependent increase in fluorescence emission that was comparable to that Figure 5. hPrP90–231 peptide incubation time course in physiological condition of cell viability (A) and membrane ionic conductance (B). hPrP90–231 was incubated at 37uC in 1.8 mM Ca++, for different times, before being used in cell viability (MTT assay) or Tip Dip experiments. The grey horizontal bar in both plots represents the range of high toxicity (Fig. 5A) and the corresponding range of high conductance (Fig. 5B) obtain treating hPrP90–231 in different conditions (see Fig. 3). The two plots show that between 15 and 24 hours of hPrP90–231 incubation there is a minimum cell viability and a maximum membrane conductance values. doi:10.1371/journal.pone.0038314.g005 induced by thermal denaturation (data not shown). Similar results were also obtained evaluating the hydrophobicity of the peptide: as observed after thermal denaturation [7], increasing [Ca++] induced a higher hydrophobic amino acid exposure on hPrP90– 231 surface as measured by TNS binding (Fig. 8A). Finally, one of the hallmarks of pathogenic PrP is proteinase K resistance, which can be induced in hPrP90–231 by thermal denaturation [7]. Incubation with Ca++ (5 and 10 mM) signifi- cantly increased the resistance of hPrP90–231 to proteinase K digestion, with the effect induced by the concentration of 10 mM comparable to that caused by incubation at 53uC for 1 hr (Fig. 9). Conversely, hPrP90–231 in native conformation, or after the incubation with low [Ca++] (1.8 mM), was completely digested even at the lowest PrP/PK ratio (500/1) (Fig. 9). Similar results were obtained using hPrP90–231 mutants. E200K mutation that per se shows high TNS binding (Fig. 8B) and tryptophan PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e38314