We have studied the effects of macromolecular crowding on protein folding kinetics by studying the oxidative refolding of hen lysozyme in the absence and presence of high concentrations of bovine serum albumin and Ficoll 70. The heterogeneity characteristic of the lysozyme refolding process is preserved under crowded conditions. This, together with the observation that the refolding intermediates that accumulate to significant levels are very similar in the absence and presence of Ficoll, suggests that crowding does not alter substantially the energetics of the protein folding reaction. However, the presence of high concentrations of macromolecules results in the acceleration of the fast track of the refolding process whereas the slow track is substantially retarded. The results can be explained by preferential excluded volume stabilization of compact states relative to more unfolded states, and suggest that, relative to dilute solutions, the rates of many protein folding processes are likely to be altered under conditions that more closely resemble the intracellular environment.

Prions arise when the cellular prion protein (PrPC) undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrPSc. Frequently, PrPSc 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. In 22 independent experiments, recPrP amyloid preparations, but not recPrP monomers or oligomers, transmitted disease to transgenic mice (n = 164), denoted Tg9949 mice, that overexpress N-terminally truncated PrP. Tg9949 control mice (n = 174) did not spontaneously generate prions although they were prone to late-onset spontaneous neurological dysfunction. When synthetic prion isolates from infected Tg9949 mice were serially transmitted in the same line of mice, they exhibited sPrPSc and caused neurodegeneration. Interestingly, these protease-sensitive prions did not shorten the life span of Tg9949 mice despite causing extensive neurodegeneration. We inoculated three synthetic prion isolates into Tg4053 mice that overexpress full-length PrP; Tg4053 mice are not prone to developing spontaneous neurological dysfunction. The synthetic prion isolates caused disease in 600–750 days in Tg4053 mice, which exhibited sPrPSc. These novel synthetic prions demonstrate that conformational changes in wild-type PrP can produce mouse prions composed exclusively of sPrPSc.

The substitution of alanines for the two cysteines which form thioether linkages to the haem group in cytochrome c 552 from Hydogenobacter thermophilus destabilises the native protein fold. The holo form of this variant slowly converts into a partially folded apo state that over prolonged periods of time aggregates into fibrillar structures. Characterisation of these structures by electron microscopy and thioflavin-T binding assays shows that they are amyloid fibrils. The data demonstrate that when the native state of this cytochrome is destabilised by loss of haem, even this highly K K-helical protein can form L L-sheet structures of the type most commonly associated with protein deposition diseases. ß

NMR techniques and 8-anilino-1-napthalenesulphonate (ANS) binding studies have been used to characterize the apo state of a variant of cytochrome c 552 from Hydrogenobacter thermophilus. In this variant the two cysteines that form covalent thioether linkages to the heme group have been replaced by alanine residues (C11A/C14A). CD studies show that the apo state contains ϳ14% helical secondary structure, and measurements of hydrodynamic radii using pulse field gradient NMR methods show that it is compact (R h , 16.6 Å). The apo state binds 1 mol of ANS/mol of protein, and a linear reduction in fluorescence enhancement is observed on adding aliquots of hemin to a solution of apo C11A/C14A cytochrome c 552 with ANS bound. These results suggest that the bound ANS is located in the heme binding pocket, which would therefore be at least partially formed in the apo state. Consistent with these characteristics, the formation of the holo state of the variant cytochrome c 552 from the apo state on the addition of heme has been demonstrated using NMR techniques.

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