Calcium Dependent Prion Protein Toxic Conformation highest concentrations to a loss of spectrophotometric signal, likely due to protein aggregation. B. CD analysis of hPrP90–231 E200K mutant in the presence of increasing concentrations of Ca++. The peptide structure, already enriched in b-sheet content, is not significantly affected by the incubation with the ion. C. CD analysis of hPrP90–231 D202N mutant in the presence of increasing concentrations of Ca++. Incubation in high Ca++ concentration reduce the a-helix content, although showing a lower sensibility than the w.t. protein. At the highest concentration tested a loss of spectrophotometric signal occurs, likely due to protein aggregation. doi:10.1371/journal.pone.0038314.g006 fluorescence (data not shown) showed a moderate further increase in these values, reaching the same values observed in the w.t. peptide (Fig. 8A). We previously reported a similar behavior also after thermal denaturation [17]. As far as secondary structure, hPrP90–231 E200K that is natively rich in b-sheet [17], after incubation in high [Ca++] no significant changes were observed as far as b-sheet content although a loss of signal was observed, likely due to protein aggregation (Fig. 6B). Again a similar protein behavior was detected in previous studies after thermal denaturation [17]. D202N mutation that mildly affected native hPrP90–231 structure [17], acquired increased hydrophobicity (Fig. 8C) and N-terminal tryptophan exposure (data not shown) after incuba- tion in high [Ca++], resembling the structural alterations Figure 6. Effect of Ca++ on secondary structure of hPrP90– 231 w.t. (A) and bearing E200K (B) or D202N (C) TSE-related mutations. A. CD analysis of hPrP90–231w.t. in the presence of increasing concentrations of Ca++ (mM: 0 = ___; 1.8 = ; 5 = ; 10 = ). A. A loss of a- helix structure and increase in b-sheet content was observed in the w.t. peptide for increasing Ca++ concentrations that was associated, at the Figure 7. Thioflavin T binding to hPrP90–231 w.t. HPrP90–231 in native, conformation, after mild thermal denaturation (536C, for 1 hr), after incubation for 1 hr in the presence of 10 mM Ca++, or after a combination of both treatments was tested for Th T binding in a fluorimetric assay, as index of b-sheet content. As previously reported, thermal denaturation increased the Th T binding as compared to the native peptide, but this effect was slightly higher after incubation with Ca++. No additivity between thermal denaturation and incubation in the presence of 10 mM Ca++ was observed. doi:10.1371/journal.pone.0038314.g007 PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e38314