Figure 8. Effect of Ca++ on hydrophobic amino acid exposure, measured by TNS binding, of hPrP90–231 w.t. (A) and bearing E200K (B) or D202N (C) TSE-related mutations in the presence of increasing concentrations of Ca++ (mM: 0 = __; 1.8 = ; 5 = ; 10 = ). As internal control, the direct effect of 10 mM Ca++ on TNS is reported (+). A. TNS binding assay of hPrP90–231 w.t after incubation with Calcium Dependent Prion Protein Toxic Conformation increasing concentrations of Ca++ show a great increase in TNS binding, indicating the increased hydrophobicity of the peptide. B. TNS binding assay of hPrP90–231 E200K showed a basal higher hydrophobicity than the w.t. peptide, although in the presence of increasing concentrations of Ca++ TNS binding greatly increased, indicating increased hydropho- bicity of the peptide C. TNS binding assay of hPrP90–231 D202N in the presence of increasing concentrations of Ca++. In these experimental conditions the hydrophobicity of the peptide greatly increased, as indicated by the TNS binding fluorescence although reaching lower levels than observed with the other peptides. doi:10.1371/journal.pone.0038314.g008 observed after thermal denaturation [17]. Interestingly, CD analysis showed that a-helix content remained unchanged in the presence of Ca++ (Fig. 6C), as was previously described after thermal denaturation [17]. Thus, to conclude this part of the study we propose that the biological effects induced by hPrP90– 231 incubation in high [Ca++] are due to a structural rearrangement that induce a conformational change closely related to the toxic conformation we previously described after mild thermal denaturation [7,17,48]. Molecular Modeling Here, the structural and electrostatic changes occurring as a result of the E200K mutation and the binding of the Ca2+ ion on the PrP90–231 structure were analysed, in comparison to the w.t. protein, by means of molecular mechanics based approaches and electrostatic potential similarity analyses. The computational protocol we used is based on the combination of LLMOD searches and multiple minimizations and allowed to develop reliable models for (i) the wild type PrP90–231 protein (model I) and its E200K mutant (model II), whose NMR structures are already well known [38,39], and (ii) the binding of the Ca++ ion by the Pr90–231 segment of PrPC (model III) that is still unknown and is the main goal of this study. The similarity between the electrostatic potential at the proteins surface was then evaluated for these models employing the Carbo´ similarity index (see Material and methods). This procedure allowed us to propose a molecular model for the binding of the Ca++ ion to PrPC protein and suggest possible structural and electrostatic alterations responsible for the increased neurotoxicity. Structural, Charge and Similarity Analyses of w.t. PrPC and the E200K Mutant Preliminarily, we evaluated the predictive performance of models I and II in reproducing the experimental behaviour of the w.t. hPrP90–231 protein and its E200K variant. Both proteins include a globular domain, residues 125–228, containing three a- helices comprising the residues 144–154 (H1), 173–194 (H2), and 200–228 (H3) and a short anti-parallel b-sheet comprising the residues 128–131 and 161–164 (Fig. 10, left panel). A comparison of the NMR structures of these two proteins shows, in spite of the Glu200RLys substitution, an almost identical overall structure, even in the vicinity of residue 200. Indeed, except for the loss of the Glu200-Lys204 salt-bridge interaction within the H3 helix and significant alterations in the highly flexible loop 167–171, only minor differences in interdomain interactions are observed. Particularly interesting is the H-bond pattern, involving the Arg156, His187 and Thr191 residues and connecting the C terminus of H1 and H2 in the w.t. protein (Fig. 10, left panel), that is lacking in the E200K variant Fig. 10, right panel). These structural differences are all nicely reproduced by our models I and II thus validating our MM approach. In spite of the almost identical three-dimensional structure, the Glu200R Lys substitution has important effects on the charge distribution at the protein surface. Indeed, the replacement of the PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e38314