Rapid folding of the prion protein captured by pressure-jump.

The conversion of the cellular form of the prion protein (PrP(C)) to an altered disease state, generally denoted as scrapie isoform (PrP(Sc)), appears to be a crucial molecular event in prion diseases. The details of this conformational transition are not fully understood, but it is perceived that they are associated with misfolding of PrP or its incapacity to maintain the native fold during its cell cycle. Here we present a tryptophan mutant of PrP (F198W), which has enhanced fluorescence sensitivity to unfolding/refolding transitions. Equilibrium folding was studied by circular dichroism and fluorescence. Pressure-jump experiments were successfully applied to reveal rapid submillisecond folding events of PrP at temperatures not accessed before.

The elusive intermediate on the folding pathway of the prion protein.

A key molecular event in prion diseases is the conversion of the cellular conformation of the prion protein (PrP(C)) to an altered disease-associated form, generally denoted as scrapie isoform (PrP(Sc)). The molecular details of this conformational transition are not fully understood, but it has been suggested that an intermediate on the folding pathway of PrP(C) may be recruited to form PrP(Sc). In order to investigate the folding pathway of PrP we designed and expressed two mutants, each possessing a single strategically located tryptophan residue. The secondary structure and folding properties of the mutants were examined. Using conventional analyses of folding transition data determined by fluorescence and CD, and novel phase-diagram analyses, we present compelling evidence for the presence of an intermediate species on the folding pathway of PrP. The potential role of this intermediate in prion conversion is discussed.

DNA vaccination can protect Cyprinus Carpio against spring viraemia of carp virus.

Several DNA constructs containing the spring viraemia of carp virus (SVCV) glycoprotein (G) gene were investigated for their ability to induce protection against SVCV following injection into myofibres. The constructs were pooled into four groups and co-injected with a plasmid encoding murine granulocyte-macrophage colony-stimulating factor. Group 1 contained one full-length and two truncated G constructs under the control of the cytomegalovirus (CMV) promoter. Group 2 contained full-length constructs with the CMV promoter, the simian virus 40 promoter and a muscle-specific promoter. Group 3 contained constructs in which the G-gene was fused with a second gene in order to improve secretion of the G-protein or to enhance destruction of transfected myocytes by T cells. Group 4 contained constructs with the CMV-Intron A promoter in plasmids with or without CpG motifs. A small-scale trial in goldfish showed that antibody responses in at least half the fish were induced by three injections of plasmids from Groups 1 and 3 whereas T-cell like responses with stimulation indices of above 3 were induced in at least half the fish by Groups 2 and 4. A single-dose of each plasmid mix was then used to protect carp in a large-scale trial. Following challenge with a heterologous strain of SVCV that killed 64% of fish, the strongest protection was observed in carp that received the full length G-gene expressed by two plasmids driven by the CMV-Intron A promoter (Group 4), with a relative percentage survival of 48% (p=0.00008).

Synthesis and structural characterization of a mimetic membrane-anchored prion protein.

During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrP(Sc)) of the host encoded prion protein (PrP(C)) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrP(C) and PrP(Sc) have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrP(C) to PrP(Sc), but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C-terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP-GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane-anchored PrP. We show that the structure of PrP-GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor-free forms of PrP represent the structure of cellular, membrane-anchored PrP. The availability of a lipid-anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP.

Stability and conformational properties of doppel, a prion-like protein, and its single-disulphide mutant.

Both prion protein and the structurally homologous protein doppel are associated with neurodegenerative disease by mechanisms which remain elusive. We have prepared murine doppel, and a mutant with one of the two disulphide bonds removed, in the expectation of increasing the similarity of doppel to prion protein in terms of conformation and stability. Unfolding studies of doppel and the mutant have been performed using far-UV CD over a range of solution conditions known to favour the alpha-->beta transformation of recombinant prion protein. Only partial unfolding of doppel or the mutant occurs at elevated temperature, but both exhibit full and reversible unfolding in chemical denaturation with urea. Doppel is significantly less stable than prion protein, and this stability is further reduced by removal of the disulphide bond between residues 95-148. Both doppel and the mutant are observed to unfold by a two-state mechanism, even under the mildly acidic conditions where prion protein forms an equilibrium intermediate with enhanced beta-structure, potentially analogous to the conversion of the cellular form of the prion protein into the infectious form (PrP(C)-->PrP(Sc)). Furthermore, no direct interaction of either doppel protein with prion protein, either in the alpha-form or the beta-rich conformation, was detectable spectroscopically. These studies indicate that, in spite of the similarity in secondary structure between the doppel and prion protein, there are significant differences in their solution properties. The fact that neither doppel nor its mutant exhibited the alpha-->beta transformation of the prion protein suggests that this conversion property may be dependent on unique sequences specific to the prion protein.