We present a complete survey of susceptibility to limited proteolysis of a PrPSc
strain (Figure S6
). The map of PK-susceptible spots: 116–118, 133–134, 141, 152–153, 162, 169, and 179, strongly suggests regions corresponding to loops and turns, while nicks at 81, 85, and 89 signal the frontier between the structured C-terminal and unstructured N-terminal domains of PrPSc
. Given the high proportion of β-sheet secondary sctructure derived from FTIR analyses, it is logical to conclude that PK- resistant stretches flanking these spots most likely are strands of β-sheet.
Our results are in excellent agreement with our previous studies of wild-type PrPSc
. Our experiments with two different SHaPrPSc
strains showed the sequence stretches 23–86 (263K), 23–101 (Dy), 117–119, 131–142, and the region around 154 (
) to be sensitive to PK. In the present study, besides confirming these regions as being PK-sensitive, we identified three additional PK cleavage sites in the C-terminal region of GPI-
We did not find evidence of any PK-resistant peptide with an N-terminus beginning beyond V179. This is not a consequence of technical limitations, since the Tricine-based SDS-PAGE allows identification of peptides as small as 3.5 kDa (). Instead, either this region is completely resistant to PK, or no stable PK-resistant cores remain if PK cleaves beyond that point.
Our results also agree with several studies describing amino-terminally truncated PK-resistant peptides in human CJD PrPSc
. Zou et al.
described human CJD PrPSc
PK-resistant C-terminal peptides spanning from positions 154/156 and 162/167 to the C-terminus 
. These fragments are analogous to GPI-
, respectively. Zanusso et al
. described two additional amino-terminally truncated human CJD PrPSc
peptides (MW of 16/17 kDa) 
, analogous to the GPI-
. Kocisko et al
. used a C-terminal antibody (epitope 217-232) to demonstrate the presence of a number of amino-terminally truncated PK-resistant species in SHaPrPSc
. Using synthetic mouse prions, Bocharova et al
. identified the regions beginning at 138/141, 152/153, and 162, and extending to the C-terminus as being resistant to PK 
. This suggests that synthetic prions and PrPSc
share key structural elements, which would explain the capacity of recombinant PrP fibrils to change their conformation, via a “deformed templating” mechanism, to that of PrPSc
In contrast, relatively few C-terminally truncated peptides have been described. Notari et al.
reported two human CJD PrPSc
peptides truncated near position 228 
. Stahl et al.
also reported the presence of a peptide truncated at position 228 in PK-treated SHaPrPSc
. The absence of such fragments in our study could be explained by slight differences in sample preparation, or perhaps by the fact that the absence of the GPI-anchor might have an effect on nearby residues.
This conspicuous absence of the C-terminally truncated peptides is a reflection of the stability of the C-terminal region, in GPI−
appears to be the most stable part of the molecule, which is inconsistent with the presence of substantial stretches of α-helical secondary structure in that region. Our results agree with Smirnovas et al
., who showed the C-terminus of GPI-
to exhibit extremely low rates of H/D exchange, typical of extensive H-bonding (β-sheet) 
. These authors showed that an FTIR absorbance band (~1,660 cm−1
) previously assigned to α-helical secondary structure in PrPSc
is also present in the spectrum of recombinant PrP amyloid fibrils, which contain no α-helices, and therefore cannot be taken as evidence of the presence of α-helical structure. They concluded that GPI−
consists of a series of β-sheet stretches connected by short loops and/or turns, in agreement with our conclusions. Some stretches exhibiting a somewhat higher exchange rate, suggested to overlap with loops/turns, such as 133–148 or 81–118, are consistent with flexible stretches identified in our study, although discrepancies also exist. The limited resolution of both analytical techniques prevents a more exhaustive comparison, but overall both of them agree.
fibrils are about 3–5 nm wide (
and our unpublished results). This constraint means that each PrPSc
monomer must be coiled in such a way as to fit approximately 140–145 residues (~G85
) into this width. To do so, PrPSc
monomers must necessarily adopt a multi-layer architecture, as seen in SH3 fibers 
or the HET-s fungal prion domain 
. The HET-s prion domain packs 70 residues into two β-strands alternating with turns and loops 
. Wille et al.
have suggested that PrPSc
fibrils are composed of four rungs of β-strands, based on their interpretation of X-ray diffraction patterns 
. In this model, each rung would comprise ~36–37 residues. Positions N152
lie near the middle of the G85
sequence, so it is tempting to speculate that they might be located at an exposed position at the border between rungs. This might explain why the N152
fragment emerges as the most conspicuous PK-resistant fragment after prolonged treatment with PK or partial unfolding with guanidine ( and ). Positions A116
might be the border between the two most amino-terminal rungs (approximately G85
). On the other hand, our results are partially inconsistent with the location assigned by Govaerts et al
., using threading algorithms, to residues K100
, placed in loops and not rungs 
. Our data show that the stretches formed by residues K100
, and Y154
, are PK-resistant, i.e
., likely part of a β-strand rung ( and ).
In summary, our data support a PrPSc structure consisting of a series of highly PK-resistant β-sheet strands interspersed with PK-sensitive short flexible loops and turns. Furthermore, the region comprising ~V179 to the C-terminus of PrPSc is probably composed primarily of β-sheet, as it is highly resistant to PK. Our data are consistent with our previous results (263K and Dy strains) and those of other researchers using SHaPrPSc. Furthermore, they are consistent with those observed for human CJD PrPSc, which suggests that the myriad human, hamster and mouse prions share a common basic structure.