Figure 1.
Alignment of Hamster and Rabbit PrP 121–231 Amino Acid Sequences.
The β2−α2 helix cap is highlighted in blue and the secondary structure locations are shown below.
Table 1.
Data collection and refinement statistics for the crystal structures of S170N, S174N and S170N/S174N mutants of rabbit PrPC 121–230
Figure 2.
Representative structure of S170N, S174N and S170/S174N mutants of rabbit PrPC 121–230.
All three structures have the typical PrP fold of three α-helices and a small two-stranded β-sheet. All three structures displayed only 0.5 Å2 root-mean-squared deviation between equivalent Cα positions when compared to each other and to wild-type. Insets: close-up views of the residues forming the helix-cap in the wild-type and their equivalents in the three mutant structures of rabbit PrPC 121–231. The reciprocal interactions between the backbone and side chains of S170 and S174 in the wild-type are ablated in the S174N and S170N/S174N mutant structures. The side chain of the mutant S170N is solvent exposed and disordered, but may weakly interact with the neighboring N171.
Figure 3.
Urea-induced unfolding curves of wild-type and helix-cap mutants of rabbit and hamster PrPC 121–230.
Samples of wild-type and helix-cap mutants of hamster and rabbit PrPC 121–230 were diluted to 10 µM in 50 mM sodium phosphate pH 7.0 with indicated concentrations of urea and incubated at room temperature for 72 hours. The proportion folded was then determined by measuring ellipticity by circular dichroism at 220 nm and normalizing between folded and unfolded baselines. A) In Hamster PrPC, the mutations of N170S and N174S caused a moderate increase in the free energy of unfolding of hamster PrPC whereas the N170S/N174S double mutation showed an additive effect. B) In Rabbit PrPC, the mutation of S170N caused a small drop in the free energy of unfolding whereas for the mutations of S174N and S170N/S174N the decrease was more significant.
Table 2.
Free energy of unfolding of wild-type rabbit and hamster PrPC 121–231 as well as S170N, S174N, S170N/S174N mutants of rabbit PrPC 121–230 and N170S, N174S, and N170S/N174S mutants of hamster PrPC.
Figure 4.
β-state propensity measurements of wild-type and helix-cap mutants of rabbit and hamster PrPC 121–230.
Samples were diluted to 10 µM in sodium acetate buffer at pH 5.0 or pH 4.5 with indicated concentrations of urea. After 72 hr incubation, the proportion of β-state was determined using the two-wavelength CD method (see Materials and Methods). (A) In hamster PrP at pH 5.0, the helix-cap mutations cause a decrease in the β-state population from 96% in the wild-type to 76%, 67%and 61% in the N170S, N174S and the N170S/N174S mutants, respectively. (B) At pH 4.5, the wild-type and helix-cap mutants all eventually populate the β-state to 100%, but to reach it they require increased urea concentrations. (C) In Rabbit PrP at pH 5.0, only the S174N and S170N/S174N mutants begin to populate the β-state to 15% and 35%, respectively. (D) At pH 4.5, the helix-cap mutations S170N, S174N and S170N/S174N cause an increase in the maximum β-state populations to 54%, 67%, and 85%, respectively, compared to 43% in the wild-type. Overall error of 4.5% was estimated from the difference between observed and fitted values.
Figure 5.
Circular dichroism wavelength scans of (A) wild-type hamster PrP 121–231, (C) hamster PrP 121–231 N170S/N174S, (B) wild-type rabbit PrP 121–230, (D) rabbit PrP 121–230 S170N/S174N.
At t = 0, samples of 100 µM PrP in 50 mM sodium acetate pH 4.0, 80 mM NaCl were diluted to a final PrP concentration of 10 µM and 4 M urea in identical buffer. Circular dichroism wavelength scans were then performed at t = 0 and t = 4 hrs between 205–250 nm at 0.1 nm intervals. The CD spectrum of α-helical PrPC is included for comparison.
Figure 6.
Time-resolved size exclusion chromatography analysis of wild-type and double helix-cap mutants of rabbit and hamster PrP.
At t = 0, samples were diluted to a final PrP concentration of 10 µM in target buffer and immediately injected onto an S200 10/30 column. Fractional concentrations of monomer and octamer were calculated; no intermediate species were detected.