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...
Membrane proteins are usually solubilized in polar solvents by incorporation into micelles. Even for small membrane proteins these mixed micelles have rather large molecular masses, typically beyond 50 000 Da. The NMR technique TROSY (transverse relaxation-optimized spectroscopy) has been developed for studies of structures of this size in solution. In this paper, strategies for the use of TROSY-based NMR experiments with membrane proteins are discussed and illustrated with results obtained with the Escherichia coli integral membrane proteins OmpX and OmpA in mixed micelles with the detergent dihexanoylphosphatidylcholine (DHPC). For OmpX, complete sequence-specific NMR assignments have been obtained for the polypeptide backbone. The 13 C chemical shifts and nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution, and in the collection of an input of conformational constraints for the computation of the global fold of the protein. For OmpA, the NMR assignments are so far limited to about 80% of the polypeptide chain, indicating different dynamic properties of the reconstituted OmpA L L-barrel from those of OmpX. Overall, the present data demonstrate that relaxationoptimized NMR techniques open novel avenues for studies of structure, function and dynamics of integral membrane proteins. ß
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