resemble the physiological composition (144 mM NaCl, 1.8 mM CaCl2, 1.2 mM MgCl2, 10 mM Hepes, pH 7.0), and the cis solution to mimic the cytoplasmic ion milieu (144 mM KCl, 0.1 mM CaCl2, 1.2 mM MgCl2, 1.1 EGTA, 10 mM Hepes, pH 6.2). Samples of hPrP90–231, wt and its E200K and D202N mutated forms employed in all Tip-Dip assays were made up to a final concentration of approximately 80 mM in a solution containing: 144 mM NaCl, 1.8 mM CaCl2, 1.2 mM MgCl2, 10 mM Hepes, pH 5.0 and DMSO 55%. 20 microliters of the stock solutions were added to 1 ml of the test solutions to a final concentration of 160 nM. In ion current measurements achieved in essaying the effect of Ca++, increasing amounts of CaCl2 (5, 10, 20, 50 mM) were added in the trans solution. Circular Dichroism (CD) Spectra were measured on a Jasco J-600 spectropolarimeter, calibrated with camphorsulphonic acid [35]. Samples were diluted in 10 mM phosphate buffer, pH 7.2. Spectra were recorded between 200 and 250 nm with a 1 nm spectral step size, 0.5 nm bandwidth and 100 nm/min scan rate, using 1 mm quartz cell. Spectra represent the average of 10 scans subtracted of blank values (10 scans of buffer alone). The experiments were independently repeated three times. Thioflavin T (Th T) Binding Assay Peptides was incubated with 10 mM Th T (Sigma-Aldrich) (in 20 mM NaOPO4, 0.15 M NaCl, pH 7.0) and fluorescence was monitored using Lambda Bio 10 spectrofluorometer (Perkin Elmer). Th T fluorescence was measured with excitation at 450 nm and emission from 460 to 560 nm with excitation and emission slits of 10 nm, as reported [36]. Spectra are reported subtracted of respective blank values. 2-(p-Toluidinyl)naphthalene-6-sulfonate (TNS) Binding Assay This assay was performed with a Spex spectrofluorimeter (model Floromax) equipped with a temperature-controlled cell compartment maintained at 18uC using the hydrophobicity fluorescent probe TNS, as reported [37]. In particular, the fluorescence emission of the complex hPrP90–231-TNS (1 mM of peptide plus 100 mM of TNS, dissolved in 2 mM phosphate buffer pH 6.0) was recorded between 400 and 510 nm, with an excitation wavelength at 365 nm. The spectra were reported subtracted of the respective blank solution containing 100 mM TNS plus 1.8, 5, or 10 mM Ca++. Intrinsic Fluorescence Measurements Fluorescence spectra were measured with a Lambda Bio 10 spectrofluorimeter (Perkin-Elmer). Tryptophan emission from of hPrP90–231 was recorded between 295 and 420 nm, with excitation at 295 nm. The spectra were reported subtracted of the respective blank values [37]. hPrP90–231 Proteinase K (PK) Resistance Assay hPrP90–231 resistance to proteolysis was revealed by immuno- blotting [7]. Digestion was performed treating 100 mg proteins with increasing concentrations of PK (1, 2, 10 mg/ml) for 30 minutes at 37uC. Digestion was stopped by boiling samples in Laemmli buffer. Digestion profile was detected subjecting samples to 15% SDS-PAGE followed by immunoblotting using the anti PrP mouse monoclonal antibody 3F4 (epitope corresponding to amino acids 109–112 in the human PrP: Signet Lab, London, UK). Calcium Dependent Prion Protein Toxic Conformation Computational Details: Structural Analyses of PrPC Wild Type and E200K Mutant The experimental NMR structures of the recombinant wild type hPrP90–230 and the hPrP90–231 E200K mutant were taken from the PDB archives, entry code 1QM0 [38] and 1FKC [39], respectively. The selected entries were graphically inspected and analyzed to find possible structural mismatches by using the molecular modelling software Maestro [40]. A geometry minimization has been preliminarily performed for both proteins, starting from the experimental structures, by using MacroModel [41] with the OPLS-AA force field [42,43] and a gradient threshold of 0.05 kJ mol21 A˚21. The water environment was simulated with the GB/SA implicit solvation model imple- mented in the program [44]. The structures so obtained correspond to local minima of the potential energy surfaces (PES) for the two proteins and were then employed as input for large-scale low mode (LLMOD) searches [45,46] allowing the characterization of further local minima in a wider section of their PES. The LLMOD calculations were carried out by imposing the default set-up to the search variables, carrying out 1000 Monte Carlo steps before stopping, setting 3.0 and 6.0 A˚ as minimum and maximum distance step, respectively, and imposing a convergence limit to the local minimization steps of 1.0 kJ mol21 A˚21, to increase the time performances of the search. The exploration of the first 30 pure normal modes correspond- ing to the eigenvectors of the input structure Hessian with the lowest frequencies was carried out by the global search option, allowing each Monte Carlo step to begin with the preceding structure, provided its energy is within 100 kJ mol–1 of the global minimum. The LLMOD outputs were subsequently minimized and characterized as unique structures within a gradient threshold of 0.050 kJ mol21 A˚21, using the multiple minimization module implemented in MacroModel. The multiple minimization outputs were further filtered to eliminate high energy conformation and, eventually, the structure corresponding to the global minimum was taken as representative of the water solution structure for each PrPC form, and hereafter referred to as models I and II. These two models were then superimposed by the minimization of root mean square deviations (RMSD) of Ca backbone positions. The resulting structures were centered and aligned with respect to center of mass and moment of inertia, using the dedicated tool of Maestro. The direction of the smallest principal moment of inertia, used as x-axis in the workspace coordinates of Maestro, is almost aligned with H3, the longest among the three helices character- izing the three-dimensional structure of PrPC. Preliminarily, the calculated structures of models I and II were graphically inspected with Maestro to retrieve qualitative infor- mation on the main differences and similarities between the two PrPC forms. In particular, we focused on the distribution of charged residues on the whole protein surface because of their major contribution to the molecular electrostatic potential (MEP). We also characterized salt bridges and most stable hydrogen bonds (involving charged residues) in terms of intra or interdomain interactions which are expected to play a major role in the stabilization of the tertiary structure. A more detailed analysis was then performed by comparing the MEPs of models I and II in the space surrounding the superimposed protein models (I U II) by means of a in-home Perl script (Fig. S1). Both MEPs were probed on the same three-dimensional grid around the (I U II) superimposed structure and characterized by a grain of 0.35 A˚ and dimensions of 66647640 A˚, large enough to ensure an adequate mapping of MEP in the space surrounding the entire PLoS ONE | www.plosone.org 13 July 2012 | Volume 7 | Issue 7 | e38314