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- Materials and methods
The prion protein occurs as a globular domain and a leading fragment whose structure is not well-defined. For the ovine species, all of the tryptophan residues are in the initial fragment, while the globular domain is rich in tyrosine residues. Using heme as a spectroscopic probe, we have studied the recombinant prion protein before and after a temperature-induced conformational change. As for most heme proteins, the absorption spectrum of heme-CO displays a red shift upon binding to the protein, and both the Y and W fluorescence are highly quenched. Flash photolysis kinetics of the PrP–heme-CO complex shows a low yield for the bimolecular phase, indicating a pocket around the hemes. By comparing the holoprotein and the truncated sequence corresponding to the globular domain, the stoichiometry was determined to be five hemes for the globular domain and two hemes for the leading fragment. At high temperature, the hemes are released; upon cooling, only two hemes bind, and only the tryptophan fluorescence is quenched; this would indicate that the globular domain has formed a more compact structure, which is inert with respect to the hydrophobic probe. The final state of polymerization is perturbed if the synthetic peptide “N3” (PrP residues 142–166, which include the first helix) is added to the prion protein solution; the temperature cycle no longer reduces the number of heme binding sites. This would indicate that the peptide may alter or inhibit the polymer formation.
The spongiform encephalopathies or “prion diseases” are neurodegenerative pathologies affecting a growing number of animal species. Sheep scrapie, bovine encephalopathy, Kuru, Gerstmann-Sträussler-Scheinker syndrome, and Creutzfeldt-Jakob disease (CJD) belong to these diseases, which may be sporadic or transmitted. Several hypotheses have been advanced concerning the nature of the infectious agents, but the most widely admitted, the “protein only” hypothesis, proposes a proteic nature of the infectious agent (Bolton et al. 1982; Prusiner 1982). According to this hypothesis, the mechanism of the pathogenesis is the conversion of the cellular form of the prion protein (PrPc) of the host, into a pathogenic isoform (PrPsc) characterized by its insolubility, its high content in β-sheet, and its protease resistance. The difference between PrPsc and PrPc is only conformational, because no covalent modifications differentiate PrPsc from PrPc (Stahl et al. 1991).
The transmission of bovine spongiform encephalopathy could then occur via protein–protein interactions (Prusiner 1982; Caughey 2001). As for HbS in sickle cell disease, a nucleus of several proteins forms a template that can deplete the stock of remaining protein by forming a larger aggregate. Unlike HbS, the reaction for PrP is not reversible, the aggregate is highly resistant to degradation, and the pathogenic form is transmissible (Baudin-Chich et al. 1990; Lesecq et al. 1996, 1997). Thus, the protein conformation is of importance to understanding the pathology of this disease.
As for most complex reactions, the intermediate forms for the transition PrPc PrPsc have not yet been isolated and analyzed. For the prion protein, even the end points are not well-described. For the nonpathologic form PrP, the structure of the globular domain (residues 104–256) was determined by Nuclear Magnetic Resonance, while the leading fragment (8–103) did not show a “stable” form (Riek et al. 1998; Lopez Garcia et al. 2000; Zahn et al. 2000). As for other protein aggregates, the pathological form PrPSc involves the formation of amyloid deposits whose molecular structure is difficult to determine. The initial events leading to deposit formation are thus of interest (Prusiner et al. 1983).
In this study we use heme as a probe of the initial conformation of the monomeric recombinant protein, and the temperature-induced polymeric form. Heme has proven to be a useful probe, because it allows detection by both absorption and fluorescence methods. Furthermore, these standard optical techniques allow a relatively simple and rapid test that is sensitive to protein conformation. In the case of calmodulin, heme binding occurs on a millisecond time scale, and heme-CO binds with high affinity only to the calcium bound conformation (Marden et al. 1990, 1994; Leclerc et al. 1993a).
We have studied both the full sequence of ovine PrP, and the truncated form, corresponding to the globular domain. The ovine sequence has the advantage that all nine of the tryptophan residues are in the initial flexible domain, allowing a partial separation of the tyrosine fluorescence from the globular domain (shaded in Fig. 1). We show that heme binds to both domains of the PrP, but the heat-induced polymer releases heme from the globular domain.
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- Materials and methods
The present spectroscopic studies show a clear difference in the binding of the hydrophobic probe to the two domains of PrP. Five molecules of heme-CO can bind to the globular domain of PrP. This part of the protein is rich in tyrosine (12 residues), which forms three clusters. One possible binding mechanism is to insert the porphyrin between the tyrosine aromatic rings. This type of stacking has been proposed for the formation of heme dimers and higher polymers. As expected, the tyrosine fluorescence is highly quenched by the hemes.
The most surprising result is the release of the hemes after the temperature cycle. Previous studies have shown that the temperature cycle induces a transition from the α-helix form to a conformation with mainly β-sheet (Rezaei et al. 2002), as for the transition PrP PrPSc. An unfolded or partially folded protein might expose additional hydrophobic surfaces that could bind the probe. However the T-cycled recombinant PrP showed no interaction of the globular domain with heme. In fact, the tyrosine fluorescence was higher than the initial form, indicating a modified environment and little quenching.
The heat-induced PrP transconformation was accompanied by a polymerization with an apparent molecular weight estimated to be 7 MD (Rezaei et al. 2002). The final form, therefore, has significant protein–protein interaction, and little space available for heme binding. A compact form, inert to external probes, would be compatible with the high resistance of the pathological form to degradation. However, it has not yet been shown that the temperature-induced aggregate has the pathogenic properties of PrPSc.
A different number of hemes bind to the two forms: before and after the T-cycle, which provokes the increase in the β-sheet conformation:
Heme as a probe is thus sensitive to the initial steps of the multimer formation, and the absorption spectrum of heme was used to detect the early steps of PrP polymer formation (Fig. 8).
We also studied here whether the peptide N3 could participate in the temperature-induced formation of PrP polymers. We observed that addition of the peptide to the heme + PrP solution changed the properties of the final state. Without the peptide N3, the T-cycle provokes the releases of hemin, as evidenced by the recovery of the tyrosine fluorescence; with the peptide the PrP did not change its capacity to bind heme, as if the peptide prevented the formation of the form inert to hemin binding. A protein fragment such as N3 could therefore be used as an inhibitor. If it possesses the structural motif sufficient for insertion into the polymer, but lacks the complementary side to continue the polymer growth, the peptide would act as a cap to the fibre and inhibit further proteins from participating (Fig. 10). The peptide N3 appears to play this role, and could thus serve as a template to develop an inhibitor to the formation of PrPSc.