The NMR structure of the recombinant elk prion protein (ePrP), which represents the cellular isoform (ePrP C ) in the healthy organism, is described here. As anticipated from the highly conserved amino acid sequence, ePrP C has the same global fold as other mammalian prion proteins (PrPs), with a flexibly disordered ''tail'' of residues 23-124 and a globular domain 125-226 with three ␣-helices and a short antiparallel ␤-sheet. However, ePrP C shows a striking local structure variation when compared with most other mammalian PrPs, in particular human, bovine, and mouse PrP C . A loop of residues 166 -175, which links the ␤-sheet with the ␣2-helix and is part of a hypothetical ''protein X'' epitope, is outstandingly well defined, whereas this loop is disordered in the other species. Based on NMR structure determinations of two mouse PrP variants, mPrP[N174T] and mPrP[S170N,N174T], this study shows that the structured loop in ePrP C relates to these two local amino acid exchanges, so that mPrP[S170N,N174T] exactly mimics ePrP C . These results are evaluated in the context of recent reports on chronic wasting disease (CWD) in captive and free-ranging deer and elk in the U.S. and Canada, and an animal model is proposed for support of future research on CWD.transmissible spongiform encephalopathy ͉ chronic wasting disease C hronic wasting disease (CWD) is a neurological disorder in cervids that has been shown to be a transmissible spongiform encephalopathy (TSE) or ''prion disease''; other prion diseases include scrapie in sheep, bovine spongiform encephalopathy (BSE), and Creutzfeldt-Jakob disease in humans (1-3). CWD was diagnosed in 1978 as a TSE in a captive mule deer (Odocoileus hemionus) (4, 5) and was diagnosed in 1981 in a free-ranging elk (Cervus elaphus nelsoni) (6). Today, CWD is known to affect captive and free-ranging elk, mule deer, and white-tailed deer (O. virginianus), and the disease seems to be endemic in areas of the western United States and Canada.CWD is unique among TSEs by the fact that is has been studied in free-ranging species (7). The natural route of exposure appears to be oral, possibly through direct interaction between animals or through environmental contamination (8, 9). Although there is evidence for transmission to different mammalian species by intracerebral inoculation (7, 10, 11), domestic animals such as cattle, sheep, and goats are not known to be naturally susceptible to CWD (12). Compared with bovine spongiform encephalopathy (BSE), CWD transmission to cattle, goats, and laboratory animals has been reported to be inefficient, suggesting that there is a rather stringent species barrier (13-15). Cell-free conversion experiments (15) led to the prediction that TSE transmission efficiency from cervids to humans might be similar to that from cattle to humans, which is clearly of serious concern. Overall, the potential threat to livestock and the human population from the recent spread of CWD in free-ranging cervids in the U.S. and Canada is still difficult to assess, and further r...

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The effects of mutation of key active-site residues (Arg-47, Tyr-51, in Bacillus megaterium flavocytochrome P450 BM3 were investigated. Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate : R47A mutant, K m 859 µM, k cat 3960 min −" ; Y51F mutant, K m 432 µM, k cat 6140 min −" ; wild-type, K m 288 µM, k cat 5140 min −" ). A slightly increased k cat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (∆G ‡ ) resulting from a smaller ∆G of substrate binding. The side chain of Phe-42 acts as a phenyl ' cap ' over the mouth of the substrate-binding channel. With mutant F42A, K m is massively increased and k cat is decreased for oxidation of both laurate (K m 2.08 mM, k cat 2450 min −" ) and arachidonate (K m 34.9 µM, k cat 14 620 min −" ; compared with values of 4.7 µM and 17 100 min −" respectively for wild-type). Amino acid Phe-87 is critical for efficient catalysis. Mutants F87G and F87Y not only exhibit increased K m and decreased k cat values for fatty acid

The NMR structures of the recombinant cellular form of the prion proteins (PrP C ) of the cat (Felis catus), dog (Canis familiaris), and pig (Sus scrofa), and of two polymorphic forms of the prion protein from sheep (Ovis aries) are presented. In all of these species, PrP C consists of an N-terminal flexibly extended tail with Ϸ100 amino acid residues and a C-terminal globular domain of Ϸ100 residues with three ␣-helices and a short antiparallel ␤-sheet. Although this global architecture coincides with the previously reported murine, Syrian hamster, bovine, and human PrP C structures, there are local differences between the globular domains of the different species. Because the five newly determined PrP C structures originate from species with widely different transmissible spongiform encephalopathy records, the present data indicate previously uncharacterized possible correlations between local features in PrP C threedimensional structures and susceptibility of different mammalian species to transmissible spongiform encephalopathies.mammalian species ͉ feline transmissible spongiform encephalopathy ͉ scrapie T he prion protein (PrP) in mammalian organisms has attracted keen interest because of its relation to a group of invariably fatal neurodegenerative diseases, the transmissible spongiform encephalopathies (TSEs) or ''prion diseases,'' which include bovine spongiform encephalopathy (BSE), CreutzfeldtJakob disease in humans, feline spongiform encephalopathy, and scrapie in sheep. It is well established that expression of the host-encoded PrP is essential for TSE propagation (1, 2). In transgenic mice lacking the gene that encodes PrP, TSEs could not be observed, and the susceptibility toward TSE of these mice could only be restored by reestablishing PrP expression (3). High sequence conservation of PrP in mammalian species (4) indicates that this protein is functionally important in the healthy organism (1, 2), but the search for this unknown function is still ongoing.PrP was identified in the context of TSEs in an aggregated ''scrapie'' isoform of PrP (PrP Sc ) (5), which copurifies with the infective agent (6). This osbservation, the apparent stability of the infectious agent under DNA͞RNA denaturing conditions (7), and the unusual progression of the disease (8) led to the ''protein-only hypothesis.'' This hypothesis proposes that the major component, if not the only one, of the infectious particle causing TSE is a protein, i.e., presumably PrP Sc (1,(7)(8)(9)). An early observation in TSE infections has been the species barrier (10). Compared with transmission with infectious material from the same species, the incubation time for onset of TSEs is prolonged if a given species is challenged with infectious brain homogenate originating from another species. The incubation time may be reduced by consecutive passages within the new host, whereby the adaptation to the new host can take several generations for the disease to show clinical signs (11). In vivo and in vitro experiments indicated that the species ba...

The NMR structures of the recombinant prion proteins from chicken (Gallus gallus; chPrP), the red-eared slider turtle (Trachemys scripta; tPrP), and the African clawed frog (Xenopus laevis; xlPrP) are presented. The amino acid sequences of these prion proteins show Ϸ30% identity with mammalian prion proteins. All three species form the same molecular architecture as mammalian PrP C , with a long, flexibly disordered tail attached to the N-terminal end of a globular domain. The globular domain in chPrP and tPrP contains three ␣-helices, one short 310-helix, and a short antiparallel ␤-sheet. In xlPrP, the globular domain includes three ␣-helices and a somewhat longer ␤-sheet than in the other species. The spatial arrangement of these regular secondary structures coincides closely with that of the globular domain in mammalian prion proteins. Based on the low sequence identity to mammalian PrPs, comparison of chPrP, tPrP, and xlPrP with mammalian PrP C structures is used to identify a set of essential amino acid positions for the preservation of the same PrP C fold in birds, reptiles, amphibians, and mammals. There are additional conserved residues without apparent structural roles, which are of interest for the ongoing search for physiological functions of PrP C in healthy organisms.nonmammalian species ͉ transmissible spongiform encephalopathy T he prion protein (PrP) has attracted a lot of interest because of its relation to transmissible spongiform encephalopathies (TSEs), which are a group of invariably fatal neurological diseases (1). Healthy organisms that do not express a prion protein, such as suitably selected transgenic laboratory animals, cannot develop a TSE (2), and the ''protein-only hypothesis'' further attributes TSE-causing infectivity to an aggregated ''scrapie form'' of PrP (PrP SC ) that has been isolated from brain tissue of diseased organisms (1). Although TSEs have only been documented for mammalian species, PrP has been identified in a wider range of higher organisms, which on an evolutionary scale extends at least down to amphibians (3-9). In apparent contrast to the high sequence conservation among mammalian PrPs, no physiological function has been reliably attributed to the ''cellular form'' of PrP (PrP C ) found in healthy organisms.In view of its critical role in TSEs, the prion protein has also attracted considerable interest by structural biologists. So far, atomic resolution structure determination was focused on recombinant mammalian prion proteins (10-18), which have recently been shown to represent the protein architecture of PrP C (19). As a group, the mammalian PrPs have Ϸ90% sequence identity (4). Here, we present the NMR structures of recombinant PrP from chicken, turtle, and frog (chPrP, tPrP, and xlPrP, respectively), each of which has Ϸ30% sequence identity with mammalian PrP. We then exploit this low homology in searches, based on comparison of the three-dimensional structures, for conserved amino acids with apparent roles in maintaining a common PrP C -fold, and for nonst...