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Results: 7

1.
Figure 6.

Figure 6. From: Octapeptide repeat insertions increase the rate of protease-resistant prion protein formation.

Second derivative ATR-FTIR spectra of the 145stop peptides show remarkably similar β-sheet architecture. Amyloid pellets in aqueous slurries were scanned from 1400–1800 cm−1, as described in Materials and Methods. The spectral peaks at 1627 and 1637 cm−1 represent the acquisition of significant β-sheet structure upon formation of amyloid from natively unstructured precursors.

Roger A. Moore, et al. Protein Sci. 2006 March;15(3):609-619.
2.
Figure 2.

Figure 2. From: Octapeptide repeat insertions increase the rate of protease-resistant prion protein formation.

Hamster 145stop peptides containing five to 15 repeats form fibrils. Samples were analyzed at 0 h (A–E) and after completion of the fibrillization reactions by 24 h (F–J). The micrographs are at 100,000 × magnification. Representative micrographs are shown. No consistent morphological differences were observed between fibrils containing five to 15 repeat motifs. Bars, 500 nm.

Roger A. Moore, et al. Protein Sci. 2006 March;15(3):609-619.
3.
Figure 1.

Figure 1. From: Octapeptide repeat insertions increase the rate of protease-resistant prion protein formation.

Purity of hamster 145stop peptides containing five to 15 octapeptide repeats. Samples were analyzed by 18% SDS-PAGE on Tris-glycine gels stained with Coomassie Blue (left panel) or Western blot using the hamster PrP-specific monoclonal antibody 3F4 at a 1:20,000 dilution (right panel). The preparations were ~99% pure by SDS-PAGE analysis. Molecular mass markers in kilodaltons are shown to the left.

Roger A. Moore, et al. Protein Sci. 2006 March;15(3):609-619.
4.
Figure 4.

Figure 4. From: Octapeptide repeat insertions increase the rate of protease-resistant prion protein formation.

Amyloid isoforms of 145stop-5 and 145stop-11 are more protease-resistant than their non-fibrillar counterparts. 145stop-5 (A) and 145stop-11 (B) peptides at a concentration of 75 μM were subjected to PK digestion as described in the Materials and Methods. The non-digested portion of each peptide was calculated from the intensity of the Western blot bands with digitizing software. Each digestion was done in triplicate. The error bars indicate the mean ± SEM. (*) P = 0.02 and (**) P = 0.007 when the protease resistance of the amyloid form was compared with the non-amyloid form using the unpaired student’s t-test. There was no significant difference in protease resistance between the amyloid forms of 145stop-5 and 145stop-11 (P > 0.2 using the unpaired student’s t-test).

Roger A. Moore, et al. Protein Sci. 2006 March;15(3):609-619.
5.
Figure 5.

Figure 5. From: Octapeptide repeat insertions increase the rate of protease-resistant prion protein formation.

Each 145stop octapeptide repeat mutant contains the same protease resistant core. Coomassie Blue stained SDS-PAGE gel showing the protease-resistant fragment obtained from the partial PK digestion of fibrillized 145stop peptides containing five to 15 copies of the octapeptide repeat region. Each peptide was digested at 100 μM concentration in either the absence (−) or presence (+) of 1 μg/ mL PK for 1 h at 37C. For each peptide, PK digestion yielded a protease-resistant fragment with the N-terminal sequence (QWNKPS…) corresponding to PK cleavage at residue 98 of hamster PrP.

Roger A. Moore, et al. Protein Sci. 2006 March;15(3):609-619.
6.
Figure 3.

Figure 3. From: Octapeptide repeat insertions increase the rate of protease-resistant prion protein formation.

Amyloid formation occurs more rapidly as the number of octapeptide repeats increases. (A) Hamster PrP 145stop peptides were vigorously agitated in 25 mM BisTris Propane (pH 7.0), and the emission spectrum of ThT at 482 nm was recorded over 18 h. A representative experiment is shown. (B) The duration of the lag phase, defined as the length of time in which no significant change in the background level of ThT fluorescence was observed, was used to compare the kinetics of fibril formation between peptides. The bars represent an average of three samples ±SEM. (*) P < 0.01 when compared with 145stop-5 as the control using 1-way ANOVA with Dunnett’s post test.

Roger A. Moore, et al. Protein Sci. 2006 March;15(3):609-619.
7.
Figure 7.

Figure 7. From: Octapeptide repeat insertions increase the rate of protease-resistant prion protein formation.

PrP-res formation and PrP-sen:PrP-res binding occur more rapidly in hamster PrP-sen with 15 copies of the octapeptide repeat. Immunoprecipitated 35S-HaPrP-sen containing five (HaPrP-5) or 15 (HaPrP-15) copies of the octapeptide repeat was incubated in the presence of HaPrP-res as described in Materials and Methods. The percentage of input 35S-labeled HaPrP-sen converted to 35S-HaPrP-res (% conversion) was determined over 24 h (A). Error bars represent the mean ± SEM for sample size N =4. (*) P =0.02 or (**) P =0.003 using the unpaired student’s t-test. HaPrP-5 (B) or HaPrP-15 (C) was incubated with hamster PrP-res in a cell-free conversion reaction. At the indicated time points, the amount of PrP-sen bound to PrP-res (black bars) and the amount of PrP-sen converted to PrP-res (gray bars) were quantified as a percentage of the input 35S-labeled HaPrP-sen. In order to correct for non-specific pelleting of radiolabeled HaPrP-5 and HaPrP-15 in the binding assay, the amount of radiolabeled PrP in the pellet in the absence of PrP-res was subtracted from the amount of radiolabeled PrP in the pellet in the presence of PrP-res. The columns and error bars show the mean ± SEM, respectively, for N =4–6 samples. The asterisksrepresentthelasttimepointatwhichtherewasnofurtherchangein the amount of PrP-sen bound to PrP-res using a 1-way ANOVA with Dunnett’s post test (**, P <0.01 when compared with the 4-h time point). For each data set at any given time point, there was no significant difference between the percentage of PrP-sen bound and the percentage of PrP-sen converted to PrP-res (P >0.1 using the paired student’s t-test).

Roger A. Moore, et al. Protein Sci. 2006 March;15(3):609-619.

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