Protease-sensitive synthetic prions.

Prions arise when the cellular prion protein (PrP(C)) undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP(Sc). Frequently, PrP(Sc) is protease-resistant but protease-sensitive (s) prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec) PrP into amyloid fibers.

Oxidative refolding of amyloidogenic variants of human lysozyme.

The oxidative refolding of human lysozyme and its two best characterised amyloidogenic variants, Ile56Thr and Asp67His, has been investigated in vitro by means of the concerted application of a range of biophysical techniques. The results show that in each case the ensemble of reduced denatured conformers initially collapses into a large number of unstructured intermediates with one or two disulphide bonds, the majority of which then fold to form the native-like three-disulphide intermediate, des-[77-95]. The slow step in the overall folding reaction involves the rearrangement of the latter to the fully oxidised native protein containing four disulphide bonds.

Photo-CIDNP NMR spectroscopy of a heme-containing protein.

There are relatively few examples of the application of photo-CIDNP NMR spectroscopy to chromophore-containing proteins. The most likely reason for this is that simultaneous absorption of light by the photosensitiser molecule and the protein chromophore reduces the effectiveness of the photochemical reaction that produces the observed nuclear polarisation. We present details of experiments performed on the air-oxidised form of a small cytochrome, from the thermophilic bacterium Hydrogenobacter thermophilus, using both the wild-type protein and apo and holo forms of a double alanine b-type mutant.

NMR analysis shows that a b-type variant of Hydrogenobacter thermophilus cytochrome c552 retains its native structure.

Conversion of Hydrogenobacter thermophilus cytochrome c(552) into a b-type cytochrome by mutagenesis of both heme-binding cysteines to alanines significantly reduces the stability of the protein (Tomlinson, E. J., and Ferguson, S.