Calcium Dependent Prion Protein Toxic Conformation Figure 9. Increased proteinase K (PK) resistance of hPrP90–231 w.t. in the presence of increasing Ca++ concentrations. hPrP90– 231 w.t. was thermally denatured or preincubated with increasing concentration of Ca++ before being subjected to PK digestion with different enzyme concentrations. Partial resistance was evaluated in Western blot experiments using the anti-PrP antibody 3F4. Left panel shows representative immunoblots, while the graph on the right depicts the densitometry analysis of three independent experiments. Data are expressed as percentage of the respective recombinant peptide input. * = p,0.05 and ** = p,0.01 vs. control values. In native conditions or in the presence of low Ca++ concentrations, the peptide showed no resistance to PK digestion. After incubation with 5 or 10 mM Ca++ a partial resistance was observed for a peptide/PK ratio of 500:1. Thermal denaturation induced a higher resistance as compared to Ca++ binding, being the immunolabeled peptide band detectable also for a peptide/PK ratio of 200:1. doi:10.1371/journal.pone.0038314.g009 negatively charged Glu with a positively charged Lys residue results in a dramatic redistribution of the surface charges introducing patches of positive potential around the beginning of helix 3, and has been suggested to facilitate the PrPC to PrPSc conversion by promoting protein-protein interactions and aggre- gation [39]. The effects of the mutation on the electrostatic properties of PrPC was then investigated in detail by analyzing the similarity between the molecular electrostatic potential (MEP) near the surface of the wild type (model I) and the E200K mutant (model II). Such an analysis was the performed by calculating the Carbo´ similarity index (sC) [47] (see Materials and Methods) between the two MEPs on the points of a grid encompassing the superimposed protein models (I U II). Moreover, because the H3 helix has been suggested to play a key role in the disease-related conformational transformation of PrPC to PrPsc in vivo [50], and is also directly involved in the considered E200K mutation and (see below) in the binding of Ca2+ ions, we have perform our similarity analysis with respect to its axis (approximately corresponding to the x-axis in the workspace coordinates of Maestro, see Computational Details). Accordingly, the grid space was partitioned into slices perpendic- ular to the x axis, and the similarity index was calculated on each Figure 10. Structure of the proposed Ca++ binding site in the wild type PrP90–231 protein. On the left the site with the residues still involved in an H-bond pattern in model I (light cyan); on the right the resulting complex after Arg156 shift and Ca++ binding in model III (orange). Hydrogen bonds (dotted red) and coordinative bonds (green) are explicitly indicated. doi:10.1371/journal.pone.0038314.g010 PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e38314