Leslie Tetteh3SBS671W1298035Molecular dynamics simulations of the unfolding of mutant prion variants at elevated temperaturesAbstractThe goal of this investigation was to analyse the unfolding pattern carried out by mutant variants of the WT prion protein as well as the WT itself at 300k and 500k. The mutant variants used were T188A/K and E219K, all of which are associated with Creutzfeldt-Jakob disease. The T188 mutants are known to increase the risk of pathogenesis, whilst E219K is known to guard against it, prolonging conversion time.It was found that several different types of partially unfolded energy states seemed to be occupied, and that these were relatively stable, causing long periods of fluctuation about a point in terms of RMSD. The most unstable mutant was found to be T188K, and there was a sharp contrast between the unfolding pattern of E219K and the T188 mutants. From the literature search there was also found to be a possible mode to formation of an initial scrapie seed under partially denaturing conditions, which could be determined by experiment.IntroductionMalconformed versions of the wild-type prion protein (PrPc), commonly known as the scrapie form (PrPSc) act as infectious agents and are the cause of a number of diseases in mammalian species such as scrapie in sheep and cattle, whilst in humans they cause Gerstman Straussler-Scheinker syndrome (GSS) and Creutzfeldt-Jakob disease (CJD). An initial scrapie seed causes a change in the conformation of the WT-peptide such that fibrillogenesis occurs; these fibrils cause tissue damage and cell death and are toxic to humans and other large mammals, eventually leading in the long-term to widespread brain damage, ataxia and death (Chiti 2009, Otvos 2002). Pathogenesis arises sporadically in approximately 85% of human disease cases; genetic inheritance accounts for around 15% of all cases; and less than 1% can be attributed to exposure to infected tissue (Lawson et al 2005).

The scrapie form of the prion protein PrPSc has been shown to contain a secondary structure of about 43% beta structure and 30% alpha helix, which is relatively insoluble to detergents whilst being only partially prone to digestion by proteinase K. In contrast the wild-type prion protein PrPC is a largely -helical structure consisting of 42% alpha-helices and 3% beta-sheet content, which is soluble in detergents and susceptible to complete digestion by Proteinase K. Digestion of PrPSc by Proteinase K leaves a 27-30kDa protease resistant core (Stahl 1993, Lawson 1995).

The WT-peptide is a sialoglycoprotein usually found bound to the cell membrane by means of a GPI anchor (Pan 1993). It is composed of two distinct sections defined as the C-terminal and the N-terminal domains. The C-terminus has been crystallised by use of NMR spectroscopy and has been found to be a globular structure composed of three alpha helices, H1 H2 and H3, and an anti-parallel beta sheet formed by beta-structures S1 and S2 which flank the H1 helix. The N-terminal region has been found to be unstructured under crystallising conditions, whilst most of the SNPs known to affect protein properties with regard to amyloid formation have been found in the C-terminal globular domain (Riek 1996, Riek 1997, Riek 1998).

PrPC is formed at an initial translated length of 253 amino acids. The GPI anchor is attached to the globular part of the protein following cleavage of a carboxy-terminal signal sequence, whilst a 23 amino-acid signal sequence in the N-terminal region of PrPC targets it to the endoplasmic reticulum for processing. Post translational modification results in a mature length of 208 amino acids for huPrPC (Otvos 2002). Following glycosylation, PrP is localized to detergent resistant microdomains of the cell membrane (Vey et al 1996). The process of fibril elongation leading to the formation of amyloid arises from a conformational change of the secondary structure within PrPC. This changes its biochemical properties and leads to oligomerization from an initial soluble PrPSc seed (Ma 2002, Baumketner 2008). The fibril formed via this process eventually elongates and becomes an insoluble fibrillar growth (Caughey 2003). The susceptibility of this fibrillar formation to pressure is thought to allow for breakage and the formation of new fibrillar seeds (Cordeiro 2004).

The sequence structure used for dynamic simulations was the C-terminal domain of the protein residues 125-228. It has been previously determined that the N-terminal region of the protein is not found within most protease resistant amyloid aggregates (Prusiner 1982), and NMR performed on HaPrP amyloid aggregates has shown that it is the C-terminal domains which associate to form the fibrils structured core (Tycko 2010). Although the N-terminal region does not affect the structure of the protein, it appears to carry out biological functions and does affect the biochemical properties of PrPC.

The N-terminal region within hPrPC contains within it four tandem copper-binding repeats with sequence (PHGGGWGQ)4 which allows it to bind Cu2+ ions with high specificity, and also to interact with other cations such as calcium within the body (Pushie 2007, Campioni 2010). This Copper-binding sequence, termed an octarepeat, is located between residues 51-91 in humans and is highly conserved amongst all mammalian species implying that it is essential to the proper function of PrPC (Rheede 2003, Mastriani 2000). X-ray crystallographic data has been obtained showing the copper bound form of the OR repeat sequence (Burns 2002).

Studies on murine deletion mutants rPrP51-90 and rPrP32-121 have shown that whilst this N-terminal region is not needed for structure, deletion does affect the stability of the protein making for faster fibrillar formation with a shorter lag phase, a higher susceptibility to pressure, and a lower unfolding temperature (Cordeiro 2005). The effect of pressure may explain why fibrillar formations are so prone to breakage, and the shorter lag phase offers support for the theory that fibril break-up leads to the initiation of new seeds (Cordeiro 2004). The amount of beta-sheet structure formed upon subjection to fibrilising conditions has also been shown to increase with the amount of the N-terminal region deleted;19% in rPrP23-231; 25% in rPrP51-90 and 27% rPrP32-121, a trait which was also expressed in the Syrian hamster recombinant rPrP90-231 (Torrent 2004). The evidence thus demonstrates that the N-terminal region of PrP protects against fibrillogenesis by preventing beta-sheet formation.

An explanation for this difference in stability after the loss of the N-terminal region can be offered by the study of cation-pi interactions within the Prion protein. The strength of cation-pi bonding within the Prion Protein has been shown to be on a par with the energies demonstrated by other non-covalent bonds such as Van Der Waal interactions, hydrogen bonding and salt bridges, measuring at energies of between 10 and 150kJ/mol (Priyakumar 2004). These electrostatic bindings are created between the electron-rich pi bonds in aromatic and other pi-bonded residues and the positive charge present on cations. On average three cation-pi bonds were found to exist within the helix regions and two within beta sheet structures (George 2013). Given that the amino-terminal region is shown to have a high valence for binding cations this may be an important finding with regard to the loss of the N-terminal regions in most fibrillar formations.

The effect of hydration and packing on the tendency of PrPc to form beta-sheets and on its natural folding stability was carried out by Cordeiro et al in 2004 and a molecular dynamics study on the same subject was carried out by Simone et al in 2005. This study demonstrated that a less hydrated PrP structure is inherently more susceptible to pressure, which also offers some explanation as to the difference in stability of the rPrP deletion mutants, which have a less hydrated structure. However the chemical treatment of the deletion mutants with denaturing agents such as urea showed no significant differences in fibrillogenesis, indicating that the chemical mode of action leading to amyloidosis still centred around the C-terminal region of the protein (Cordeiro 2005).

The C-terminal region of PrPC is very well conserved amongst mammalian species and it has previously been found that the vast majority of single nucleotide polymorphisms which lead to genetic inheritance/resistance against prion diseases are located within the C-terminus (Billeter 1997). This carries within it the inherent notion that initiation sites for conversion to PrPSc are likely to be found within this region, whilst the high level of conservation across mammalian species implies that these initiation sites are likely to be similar in different forms of PrPC.

Point mutations that are known to lead to increased likelihood of pathogenesis are

GSSP102L, P105L, A117V, G131V, Y145X, F198S, D202N, Q217R, M232T

CJDD178N, V180N, T183A, T188A/K/R, E200K, R208H, V210I

Whilst E219K and M129V are examples of mutations that lead increased resistance to sCJD.

As can be seen in the case of GSS and CJD, within the field of Prion diseases or transmissible TSEs there are noticeable strain types, which can be identified by the incubation period; time between first exposure and development of symptoms, and the pattern of lesions which develops within the brain (Bruce 2003). Polymorphisms can account for some of the difference, but it has been shown in the studies of different clinical phenotypes of human TSEs, sCJD and sporadic Fatal Familial insomnia, that other variances are also present. Western blotting of the two aforementioned PrPSc types exposed differences that suggested glycan heterogeneity (Pan et al 2001).

The human prion protein has two asparagine linked glycosylation sites at 181N and 197N, which allow mannose (a C2 epimer of D-glucose) based oligosaccharide chains to be attached to the protein (Vey 1996). The WT peptide can be either un-, mono-, or diglycosylated when found in nature and experiments carried out by Rudd et al in 1999 on Syrian hamster PrPc and PrPSc identified more than 50 different polysaccharide chains which can be attached to the two sites. The unique glycoform ratio encountered in hPrPSc first provided the link between variant CJD and Bovine Spongiform Encephalopathy (Lawson 2005).

The differences encountered in the conformations of the Prion protein based on the source of the PrPSc offers some support for the protein-only propagation theory first expounded upon by Alper in 1967, however as evidenced by laboratory testing the type of strain produced also appears to be dependent on the available pool of PrPc. It was shown by Priola and Lawson in 2001 that the introduction of a species specific residue into muPrPC induced a conformational change that led to proteinase K resistance only with unglycosylated muPrPC. Korth et al also demonstrated in 2000 that mutations in the glycosylation consensus sequence resulted in a change in hPrPC distribution pattern within cells containing ScN2a. This alteration affects the pattern of hPrPSc formation.

It has also been demonstrated via treatment with tunicamycin, an inhibitor of N-linked glycosylation in eukaryotic organisms, and the alteration of the 181N and 197N consensus sites that unglycosylated PrPC molecules have been found that reproduce some of the characteristics of PrPSc such as detergent insolubility and partial proteinase K resistance (Esko 2009, Lehmann 1997). These studies provides some evidence that glycosylation confers some resistance to the formation of the scrapie form, which further supports the model of templated assembly put forth in the protein-only model (Prusiner 1982).

The post-translational modifications which are carried out on the C-terminal region of the protein such as glycosylation, cleavage and attachment of a GPI anchor have been demonstrated to not significantly affect the structural dynamics of PrPC in silico. The lack of impact of the N-terminal region on the NMR derived crystalline structure of the protein, and the lack of an observable effect that these modifications have further enables the sole use of the C-terminal globular region for molecular dynamics simulations. It should however be noted that oligomerization of the Prion Protein arises from different pathways, which allows for distinct amyloid formations from the same initial structure.

Kinetic partitioning has been put forward as a way of explaining amyloid formation via different transition pathways (Harrison et al 2001). This process was modelled dynamically by Dima and Thirumalai in 2002 via the use of a three-dimensional beaded lattice model containing a limited number of distinct monomer conformations. Aspects of this model showed the progression to aggregated states through the occupation of a state other than U (representing complete unfolding) or N (the native structure of the lattice) indicating that partially structured intermediates were able to form aggregated states, without necessarily having to undergo full unfolding. This model also highlighted that aspects of the aggregation process were dependent on several physiological factors such as temperature, pressure, concentration and the isoelectric point (pI) of salts in solution.

The effect of physiological factors on the unfolding characteristics of the prion protein have been greatly exploited by computer modellers. Under normal conditions for the WT-peptide, unfolding is very hard to simulate, given the tendency of the protein to occupy a minimised energy state which corresponds to its native structure under biological conditions (Daggett 2002, Rathore 2004). Altering physiological factors such as temperature, pH and hydrodynamics has been shown in some cases to greatly accelerate the unfolding speed of the prion protein and put it within a time scale that can be effectively simulated (Gu 2003).

In the present simulation the use of higher temperatures were used to induce unfolding of the prion protein in a way that highlighted potential partially structured intermediates, which can arise from protein unfolding and misfolding. The mutations chosen were T188A, T188K, and E219K, as examples of mutations known to have a varied effect on the pathogenesis of the inherited prion diseases Creutzeldt-Jakob Syndrome. The hypothesis was that the mutants would show differences in unfolding which related to their tendency to form aggregated disease states. This is highest in T188 variants and lowest in E219K.

The protein structures used for simulations were based on the NMR derived structure of the major prion protein 1QLX.

Methods

The model for the C- terminal globular domain of the WT peptide 1QLX was used. The model was downloaded from the protein database and the structures for the C-terminal fragment 125-228 were constructed using Modeller9.13. Initially modelled were the structures for naturally occurring mutant variants T188A, T188K, E219K, G142S, and N171S, which were constructed by single amino acid substitution their structures. The FASTA sequences for the C-terminal fragments are shown below. WT Peptide(125-228)LGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQR

T188A(125-228)LGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHAVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQR

T188K(125-228)LGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHKVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQR

E219K(125-228)LGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYKRESQAYYQR

MD simulations were carried out using the OPLS-AA/L all-atom force field and the GROMACS-4.6.3 package. Two hundred steps of steepest descent method was used to minimize the structure. Solvation was carried out with 3 Cl- ions. At temperatures of 300 K and 500 K the modelling was carried out using the NVT ensemble. Equilibration for 200 ps of solute position was carried out using RMD. All mutations were subject to UMD for 2 ns using the LINCS algorithm and a pH of 7 was used for the simulations. The time step used for each system was 2 femtoseconds. Three runs were carried out for each variant. RMSDs were calculated for each variant on each runstructure were calculated. Determination of the distribution of secondary structure was carried out using DSSP.

Results

Fig 1: (a-c) C RMSD values for E219K runs 1-3, (d-f) DSSP generated secondary structures for E219k runs 1-3)

Fig 2: (a-c) C RMSD values for T188A runs 1-3, (d-f) DSSP generated secondary structures for T188A runs 1-3

Fig 3: (a-c) C RMSD values for T188K runs 1-3, (d-f) DSSP generated secondary structures for T188K runs 1-3

Fig 4: (a-c) C RMSD values for WT-Peptide runs 1-3, (d-f) DSSP generated secondary structures for WT-Peptide runs 1-3

The data gained from plotting the C root mean square derivative (RMSD) values over time suggests a progressive unfolding pattern, whereby several relatively stable partially unfolded states appear to occupy several local energy minima. The unfolding of the native protein at 300k was carried out over the period of 1 nanosecond and shows the local minimum occupied by the normal structure of the wild type protein. This state appears to be between 0.25 and 0.35nm in terms of the RMSD, and all three repeats show consistency in the time taken to reach this state.Once restraints are lifted at t=0, most RMSD values of proteins at 520k rise steeply until they reach an RMSD of roughly 0.3nm, which corresponds to the structure of the prion protein in its native form. From this point there seem to be several different types of unfolding characteristics which correspond to several different stable unfolded intermediates. The highest mutant variants A local minimum energy state representing a partially unfolded intermediate is evidenced by a very slow rise in RMSD value along with a long fluctuation period between 0.4-0.5nm which is characterised by very little loss of secondary structure, instead consisting of a partially disordered state where most of the helical structure of helices H1, H2 and H3 is retained, with only slight fraying around the edges. This local minimum state can be observed in practically all of the prion variants but is particularly visible with a longer residence time in E219K runs 1+2 (fig.1a + b), T188A run 3 (fig 2c.) and T188K runs 2+3 (fig. 3b + c).Interestingly the native run at 520k seems to occupy a lower energy minimum for a longer period of time, corresponding to the native structure of the protein, maintaining a RMSD value of below 0.4nm for around 1ns in 2 out of 3 of its runs (fig 4a + c), perhaps indicating a greater stability, or the slight difference in structure already caused by the polymorphisms of the mutant variants.At an RMSD value of around 0.6nm there seems to be another local energy minimum in which resides a stable partially unfolded intermediate, characterised by a greater loss of helical character in helices H2+H3. This is particularly evidenced by E219K runs 1+3 (fig 1a + c), T188A runs 1+2 (fig 2a + b), T188K run 2 (fig. 3b) and the native run 2 (fig 4b.). Interestingly T188A runs 1+2 seem to largely bypass or have a very short residence time in the first local minimum state progressing very quickly to the greater loss of helical structure associated with the local minimum at around 0.6nm. This is particularly evident with the almost immediate disintegration of helix H2 in T188A run 2.Although the run times for unfolding were cut short at 2ns it appears as though there may also be another local energy minimum between 0.70.8nm, evidenced by T188A run 1 (fig 2a) and T188K run 1 (fig 3a), which is characterised by a loss of helical structure for helices H1+H2+H3, and appears to be differentiated from the other unfolding structures of the same variants (see figs 2b, 2c, 3b, + 3c) by the greater loss of helical content in helices H1 and H3. All prion simulations conducted at 520K showed some level of unfolding compared to the WT-peptide at 300k with all helices H1 H2+ H3 showing some level of structural change in one or more of the simulations and no obvious elongation of the beta sheets S1 and S2 or formation of beta-sheet character.DiscussionMost of the mutations associated with prion disease can be found in helices H2 and H3. This is the case of the T188A and T188K mutations found within the H2 helix, and with the E219K polymorphism found in helix H3. These two mutations are known to have contrasting effects on the mechanism of disease state formation, with T188A/K mutations known to be pathogenic and E219K having been demonstrated to protect against disease states.

The RMSD values of E219K and the T188K can be compared directly to contrast the differences in their stability. Whilst the highest RMSD values in the T188 variants can be seen to top 0.9nm and 0.8nm for T188K and T188A (fig 2a, fig 3a) respectively the highest RMSD value in the E219k is only at around 0.7nm (see fig 1.). This value obtained for the E219K variant also falls below the highest RMSD values obtained in the WT peptide which also obtains peak values at above 0.8nm.

Interestingly the E219K variant runs showed significant unfolding of the H2 helix with very little helical character seeming to remain in runs 1 and 3 (fig. 1d + f) of the unfolding process. This showed significant congruence with the possible energy minimum exhibited at RMSD of 0.6 nm, with the loss of helical character seeming to coincide with the reaching of this point.

The mechanism of inhibition of E219K is a currently under discussion. A recent 2012 study carried out by Biljan et al found that, whilst the Lysine polymorphism did not interfere with the stabilizing influences around it, it did introduce new structural features into the protein, and the replacement residue showed evidence of reduced backbone flexibility. As Lysine in solution exhibits considerably different character to that of Glutamic acid, being a basic residue as opposed to one that is positively charged, this is not an unexpected result.

It is also known that it is the heterozygous polymorphism of E219K which results in the most resistance to amyloid formation with the murine E219K variant only being shown to exhibit resistance to scrapie formation when the gene expressed is heterozygous. The reason for this is not currently known but it was also speculated by Biljan that this may be due to the formation of a dimer complex between Glutamate and Lysine 219 variants. Interestingly another polymorphism associate with resistance to prion disease the M129 mutation was shown in the cysteine variant to increase stability via an extension of the natural beta-sheet.

It is also notable that the T188K variant achieves the highest RMSD value amongst any of the simulations undertaken (fig. 3a). This again is consistent with the pathogenic nature of this mutation, which would imply a loss of stability within the protein structure. This mutation results from the replacement of threonine which in nature is a polar amino acid with Lysine which is a charged residue. This change in character is known to create new electrostatic interactions within the protein, which would lead to an alteration in stability.

It has been shown by prior studies that the T188 mutation is known to trigger flexibility and displacement of H1, but it was shown in our study, that the relatively stable fluctuation at 0.6nm is evidenced across the board, and a similar breakdown primarily in the H2 and H3 helices was observed. At an RMSD value of 0.7-0.8nm there is however shown to be a considerable breakdown of Helix H1 in both T188A and T188K variants (fig. 2d + 3d). Although there are other H1 shifts within the mutants variants, this is the only one that appears to correspond to a particular energy minimum and correspond to a stable unfolded intermediate. This would have to be corroborated by repeat testing

With regards to the secondary structure of the protein it is can also be seen that upon reaching an RMSD level of 0.6nm there is a congruent level of unfolding in the H2 helix. This is evidenced by T188K runs 2+3 (fig 3e + 3f). Similar levels of correspondence between the reaching of an RMSD value of around 0.6nm and loss of helical character in H2 can be seen as previously mentioned in T188A run 2 (fig 2e).

The understanding of the unfolding process is crucial to the understanding of the mechanism of the formation of oligomers from PrPc and proteins in general. This is because investigative work done beforehand shows that fibril formation occurs fastest at denaturing conditions, indicating that a denatured or partially denatured protein is essential to the formation of the amyloid complex (Lawson 2005).

It is often common under constraints similar to those provided here for proteins to become trapped in local minimum energy states (Tang 2012). This is a natural tendency of proteins and can be exploited to find potential characteristics of partially unfolded states. Whilst it has become generally accepted that partially structured intermediates are precursors in fibril formation, there is a lot of contrasting evidence suggesting one pathway or the other to amyloid formation (Abedini 2009).

The main point of contention in the literature around the mechanics of prion formation appears to be which structure is most unstable and prone to misfolding within the prion protein thus leading to scrapie formation. The vast range of physiological factors that can be altered in the modelling process has exacerbated this point of contention, with some studies favouring helix H1 as the most unstable structure within the prion protein, and others generally favouring a combination of helices H2 and H3 (Langella 2004, Guo 2011).

In the data provided by the DSSP view of the secondary structure it is apparent that loss of helical character in H1 is almost non-existent in most of the simulations undertaken, and in the case of run 1 for T188A and T188K does not appear to occur until the RMSD value is approximately around 0.8nm. This is in agreement with some studies which posit that a loss of helical character in H1 is unlikely to occur in the partially structured intermediate before in amyloid formation, whilst it contradicts other studies which posit that the unfolding of the H1 helix is the most populated state at melting point (Hosszu 2010, Tang 2012).

An explanation for this incongruity is offered by Garrec et al. in a recent paper published in 2013. Within this it is demonstrated by use of molecular dynamics simulations that at a pH of around 4.5 the slightly buried residue H187 assumes a protonated state which can cause one of two changes to take place in the prion protein via interaction with the linked residue R136. The repulsion caused between the protonated imidazole ring and the guanidium group on R136 appears to cause either a conformation where R136 or H187 is pushed out of place, disrupting the native conformation of the WT peptide.

The residues H187 and R136 correspond to 2 different parts of the prion protein; H187 being part of the H2 helix and R136 being located in the join between S1 and H1. Displacement of R136 from its natural cavity was shown to cause the relaxation of the S1-H1 backbone constraint and the consequent close association of regions S1-H1 and S2-H2 resulting in an elongation of the beta sheet in this region. The displacement of H187 from its cavity conversely causes a loss of intra-helical stability by repulsion with residue T183. Though both of these actions result in the loss of helical character from H2, the R136(out) populated state results in H1 destabilization as well as in the H2 helix, whilst the H187(out) populated state results in a loss of character from only the H2 helix

This result may hold implications for the possible minima observed in the T188A/K variants with T188 being very closely related within the structure, which similarly seems to display 2 states, one in which there is only a loss of helical character from Helices H2 + H3, and a second at 0.8nm where a loss of helical character is found in the H1 helix as well.

This finding may also be of importance in relation to the general process of prion formation. It is noted by Garrec et al. that the ph level of 4.5 falls within the range of physiological pH within the endosome. The transmembrane from of PrPc is found in two topological isoforms where either the C-terminal end or the N-terminal portion of the protein is directed towards the inside of the cell and the endoplasmic reticulum known respectively as CtmPrP and NtmPrP. Only CtmPrP has been implicated with any form of prion disease.

PrPC is known to be subjected to endoproteolytic cleavage which results in the production of an N-terminal and C-terminal fragment, which is then either subjected to endocytosis or enzymatically removed from the cell membrane. Marijanovic et al in 2009 demonstrated that the conversion of PrPc appears to take place within endosomal compartments, and it has been demonstrated by use of computer modelling that the H2H3 region alone can be converted to a rich form, as was remarked upon earlier in this paper with the example of the N-terminally truncated mutants rPrP51-90 and rPrP32-121 fibrils produced by Cordeiro et al. The unglycosylated form of protein as reported earlier also lowers resistance to scrapie formation, and was shown to reproduce detergent insolubility and protease resistance by Lehmann et al 1997. This makes it a greater possibility that an initial scrapie seed would be formed from an unglycosylated form of this terminal fragment, via the loss of stability in the structure and initial unfolding generated from the effect of the protonated residue H187.

Given the information provided by in this paper it appears likely to this writer that these effects, or a combination thereof, may form a possible pathway for formation of the an initial scrapie seed from PrPc. This would need to be tested by use of all the factors elucidated here which are an unglycosylated, C-terminal fragment produced by cleavage at residues 110-111, subjected to endosomal pH levels of around pH 4.5-5. From the evidence contained within this paper this writer theorizes that this would likely lead to an amyloid formation with a very similar character to PrPSc including protease resistance and detergent insolubility.

This process could be accelerated by use of phosphatidylethanolamine or PE, a common membrane protein shown to act in concert with an initial scrapie seed to increase the rate of amyloid formation by a factor of 105 (Deleault 2012). As scrapie formation is characterised by a long incubation time, this may increase the rate of reaction enough to be viewed in a suitable frame of time. Pathogenic mutations associated with disease could also be incorporated into testing to establish this.

Conclusion

From the effects seen within this paper possible supporting evidence was used to show a difference in stability between E219K and the T188 mutants. It was also shown that the proteins seem to occupy partially structured intermediates which correspond to local minimum energy states at denaturing conditions. It was also concluded from a search of the literature that it may be possible to reproduce an amyloid with all the characteristics of an initial scrapie seed under physiological conditions. This has the potential to be laboratory tested with different polymorphisms.