Prions are composed largely, if not entirely, of prion protein (PrPsc in the case of scrapie). Although the formation of PrPs from the cellular prion protein (PrPc) is a post-translational process, no candidate chemical modification was identified, suggesting that a conformational change features in PrPsc synthesis. To assess this possibility, we purified both PrPC and PrPsc by using nondenaturing procedures and determined the secondary structure ofeach. Fourier-transform infrared (FTIR) spectroscopy demonstrated that PrPC has a high a-helix content (42%) and no (3sheet (3%), findings that were confirmed by circular dichroism measurements. In contrast, the -sheet content of PrPSc was 43% and the a-helix

Variations in prions, which cause different incubation times and deposition patterns of the prion protein isoform called PrP(Sc), are often referred to as 'strains'. We report here a highly sensitive, conformation-dependent immunoassay that discriminates PrP(Sc) molecules among eight different prion strains propagated in Syrian hamsters. This immunoassay quantifies PrP isoforms by simultaneously following antibody binding to the denatured and native forms of a protein. In a plot of the ratio of antibody binding to denatured/native PrP graphed as a function of the concentration of PrP(Sc), each strain occupies a unique position, indicative of a particular PrP(Sc) conformation. This conclusion is supported by a unique pattern of equilibrium unfolding of PrP(Sc) found with each strain. Our findings indicate that each of the eight prion strains has a PrP(Sc) molecule with a unique conformation and, in accordance with earlier results, indicate the biological properties of prion strains are 'enciphered' in the conformation of PrP(Sc) and that the variation in incubation times is related to the relative protease sensitivity of PrP(Sc) in each strain.

X-ray or NMR structures of proteins are often derived without their 1 Departments of Cellular and ligands, and even when the structure of a full complex is available, the area Molecular Pharmacology and of contact that is functionally and energetically significant may be a Medicine and 2Department of specialized subset of the geometric interface deduced from the spatial Pharmaceutical Chemistry University of California proximity between ligands. Thus, even after a structure is solved, it remains a major theoretical and experimental goal to localize protein San Francisco functional interfaces and understand the role of their constituent residues. CA 94143-0450, USAThe evolutionary trace method is a systematic, transparent and novel predictive technique that identifies active sites and functional interfaces in proteins with known structure. It is based on the extraction of functionally important residues from sequence conservation patterns in homologous proteins, and on their mapping onto the protein surface to generate clusters identifying functional interfaces. The SH2 and SH3 modular signaling domains and the DNA binding domain of the nuclear hormone receptors provide tests for the accuracy and validity of our method. In each case, the evolutionary trace delineates the functional epitope and identifies residues critical to binding specificity. Based on mutational evolutionary analysis and on the structural homology of protein families, this simple and versatile approach should help focus site-directed mutagenesis studies of structure-function relationships in macromolecules, as well as studies of specificity in molecular recognition. More generally, it provides an evolutionary perspective for judging the functional or structural role of each residue in a protein structure.

Recombinant mouse prion protein (recMoPrP) produced in Escherichia coli was polymerized into amyloid fibrils that represent a subset of β sheet–rich structures. Fibrils consisting of recMoPrP(89–230) were inoculated intracerebrally into transgenic (Tg) mice expressing MoPrP(89–231). The mice developed neurologic dysfunction between 380 and 660 days after inoculation. Brain extracts showed protease-resistant PrP by Western blotting; these extracts transmitted disease to wild-type FVB mice and Tg mice overexpressing PrP, with incubation times of 150 and 90 days, respectively. Neuropathological findings suggest that a novel prion strain was created. Our results provide compelling evidence that prions are infectious proteins.

We present a Bayesian statistical analysis of the conformations of side chains in proteins from the Protein Data Bank. This is an extension of the backbone-dependent rotamer library, and includes rotamer populations and average , y angles for a full range of 4,+ values. The Bayesian analysis used here provides a rigorous statistical method for taking account of varying amounts of data. Bayesian statistics requires the assumption of aprior distribution for parameters over their range of possible values. This prior distribution can be derived from previous data or from pooling some of the present data. The prior distribution is combined with the data to form the posterior distribution, which is a compromise between the prior distribution and the data. For the ,y2. , y 3 , and ,y4 rotamer prior distributions, we assume that the probability of each rotamer type is dependent only on the previous , y rotamer in the chain. For the backbone-dependence of the , y I rotamers, we derive prior distributions from the product of the +dependent and +-dependent probabilities. Molecular mechanics calculations with the CHARMM22 potential show a strong similarity with the experimental distributions, indicating that proteins attain their lowest energy rotamers with respect to local backbone,side-chain interactions. The new library is suitable for use in homology modeling, protein folding simulations, and the refinement of X-ray and NMR structures.

Several sporadic and genetic diseases are caused by protein misfolding. These include cystic fibrosis and other devastating diseases of childhood as well as Alzheimer's, Parkinson's and other debilitating maladies of the elderly. A unified view of the molecular and cellular pathogenesis of these conditions has led to the search for chemical chaperones that can slow, arrest or revert disease progression. Molecules are now emerging that link our biophysical insights with our therapeutic aspirations.

process whereby a portion of its ␣-helical and coil structure is refolded into ␤ sheet (Pan et al., 1993). This struc- ‡ Department of Pathology § Department of Molecular and Cellular Pharmacology tural transition is accompanied by profound changes in the physicochemical properties of the PrP. While PrP C

Studies using low-resolution fiber diffraction, electron microscopy, and atomic force microscopy on various amyloid fibrils indicate that the misfolded conformers must be modular, compact, and adopt a cross-␤ structure. In an earlier study, we used electron crystallography to delineate molecular models of the N-terminally truncated, disease-causing isoform (PrP Sc ) of the prion protein, designated PrP 27-30, which polymerizes into amyloid fibrils, but we were unable to choose between a trimeric or hexameric arrangement of right-or left-handed ␤-helical models. From a study of 119 all-␤ folds observed in globular proteins, we have now determined that, if PrP Sc follows a known protein fold, it adopts either a ␤-sandwich or parallel ␤-helical architecture. With increasing evidence arguing for a parallel ␤-sheet organization in amyloids, we contend that the sequence of PrP is compatible with a parallel left-handed ␤-helical fold. Left-handed ␤-helices readily form trimers, providing a natural template for a trimeric model of PrP Sc . This trimeric model accommodates the PrP sequence from residues 89 -175 in a ␤-helical conformation with the C terminus (residues 176 -227), retaining the disulfide-linked ␣-helical conformation observed in the normal cellular isoform. In addition, the proposed model matches the structural constraints of the PrP 27-30 crystals, positioning residues 141-176 and the N-linked sugars appropriately. Our parallel left-handed ␤-helical model provides a coherent framework that is consistent with many structural, biochemical, immunological, and propagation features of prions. Moreover, the parallel left-handed ␤-helical model for PrP Sc may provide important clues to the structure of filaments found in some other neurodegenerative diseases.