Teresa J. T. Pinheiro

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.

We have developed an easy and flexible synthetic methodology to obtain lipid chains containing methanothiosulfonate terminal groups with the aim to attach them to natural proteins as functional groups. There are many proteins found in nature that are modified by lipids, and this is a key part of their function. For example, the prion protein is attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor, and this protein is thought to be the causative agent in diseases such as bovine spongiform encephalopathy (BSE; “mad cow disease”) and the human equivalent Creutzfeldt–Jakob disease. However, production of large amounts of protein in bacteria results in proteins that lack these lipid modifications. The lipid chains containing methanothiosulfonate terminal groups that we have synthesized here can be attached to these proteins through the thiol contained in the side chain of the cysteine residue, which can be incorporated into the protein sequence at the desired position.

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.

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.