Rickettsia typhi, the causative agent of murine typhus, is an obligate intracellular bacterium with a life cycle involving both vertebrate and invertebrate hosts. Here we present the complete genome sequence of R. typhi (1,111,496 bp) and compare it to the two published rickettsial genome sequences: R. prowazekii and R. conorii. We identified 877 genes in R. typhi encoding 3 rRNAs, 33 tRNAs, 3 noncoding RNAs, and 838 proteins, 3 of which are frameshifts. In addition, we discovered more than 40 pseudogenes, including the entire cytochrome c oxidase system. The three rickettsial genomes share 775 genes: 23 are found only in R. prowazekii and R. typhi, 15 are found only in R. conorii and R. typhi, and 24 are unique to R. typhi. Although most of the genes are colinear, there is a 35-kb inversion in gene order, which is close to the replication terminus, in R. typhi, compared to R. prowazekii and R. conorii. In addition, we found a 124-kb R. typhi-specific inversion, starting 19 kb from the origin of replication, compared to R. prowazekii and R. conorii. Inversions in this region are also seen in the unpublished genome sequences of R. sibirica and R. rickettsii, indicating that this region is a hot spot for rearrangements. Genome comparisons also revealed a 12-kb insertion in the R. prowazekii genome, relative to R. typhi and R. conorii, which appears to have occurred after the typhus (R. prowazekii and R. typhi) and spotted fever (R. conorii) groups diverged. The three-way comparison allowed further in silico analysis of the SpoT split genes, leading us to propose that the stringent response system is still functional in these rickettsiae.

Although de novo protein design is an important endeavor with implications for understanding protein folding, until now, structures have been determined for only a few 25-to 30-residue designed miniproteins. Here, the NMR solution structure of a complex 73-residue three-helix bundle protein, ␣ 3 D, is reported. The structure of ␣ 3 D was not based on any natural protein, and yet it shows thermodynamic and spectroscopic properties typical of native proteins. A variety of features contribute to its unique structure, including electrostatics, the packing of a diverse set of hydrophobic side chains, and a loop that incorporates common capping motifs. Thus, it is now possible to design a complex protein with a well defined and predictable three-dimensional structure.

PrP106 -126, a peptide corresponding to residues 107-127 of the human prion protein, induces neuronal cell death by apoptosis and causes proliferation and hypertrophy of glia, reproducing the main neuropathological features of prion-related transmissible spongiform encephalopathies, such as bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. Although PrP106 -126 has been shown to form amyloid-like fibrils in vitro, their structural properties have not been elucidated. Here, we investigate the conformational characteristics of a fibril-forming fragment of the mouse prion protein, MoPrP106 -126, by using electron microscopy, CD spectroscopy, NMR-detected hydrogen-deuterium exchange measurements, and molecular dynamics simulations. The fibrils contain Ϸ50% ␤-sheet structure, and strong amide exchange protection is limited to the central portion of the peptide spanning the palindromic sequence VAGAAAAGAV. Molecular dynamics simulations indicate that MoPrP106 -126 in water assumes a stable structure consisting of two four-stranded parallel ␤-sheets that are tightly packed against each other by methyl-methyl interactions. Fibril formation involving polyalanine stacking is consistent with the experimental observations. P rions are infectious particles that cause transmissible spongiform encephalopathies in animals and humans. Prions are composed of PrP Sc , a conformationally altered form of a hostencoded glycoprotein, PrP C (1). Although the two isoforms are chemically identical, they possess very different physicochemical properties. In particular, PrP C is mostly helical, whereas the scrapie form PrP Sc contains Ϸ40% ␤-sheet (2). A synthetic peptide, PrP106-126, was shown to aggregate into proteaseresistant amyloid fibrils and induce neuronal cell death by apoptosis, causing proliferation and hypertrophy of cultured glia (3, 4). This segment corresponds to an unstructured region just outside of the globular C-terminal domain of PrP C (5, 6). Analysis of deletion mutants of human prion protein (PrP) showed that a large N-terminal fragment (residues 23-88) and a segment within the structured domain of PrP C (residues 141-176) could be deleted without affecting its conversion into the protease-resistant PrP Sc , whereas deletion of segments 95-107, 108-121, or 122-140 abolished the conformational transition (7). PrP106-126 is located within this critical region (residues 95-140), has been shown to adopt different secondary structures under different solution conditions (8, 9), and is thus a relevant model for investigating the mechanism of fibril formation and PrP Sc -mediated cell death. Recent solid-state NMR results showed that fibrils of the mouse prion peptide 89-143 are composed predominantly of ␤-structure, and they suggested that its pathogenicity is related to the specific ␤-sheet conformation (10, 11). However, little is known presently about the detailed structure of PrP Sc or any fibril-forming fragments of the PrP. In fact, there are very few direct experimental observations available for any...