The NMR structures of the recombinant human prion protein, hPrP(23-230), and two C-terminal fragments, hPrP(90 -230) and hPrP(121-230), include a globular domain extending from residues 125-228, for which a detailed structure was obtained, and an N-terminal flexibly disordered ''tail.'' The globular domain contains three ␣-helices comprising the residues 144 -154, 173-194, and 200 -228 and a short anti-parallel ␤-sheet comprising the residues 128 -131 and 161-164. Within the globular domain, three polypeptide segments show increased structural disorder: i.e., a loop of residues 167-171, the residues 187-194 at the end of helix 2, and the residues 219 -228 in the C-terminal part of helix 3. The local conformational state of the polypeptide segments 187-193 in helix 2 and 219 -226 in helix 3 is measurably influenced by the length of the N-terminal tail, with the helical states being most highly populated in hPrP(23-230). When compared with the previously reported structures of the murine and Syrian hamster prion proteins, the length of helix 3 coincides more closely with that in the Syrian hamster protein whereas the disordered loop 167-171 is shared with murine PrP. These species variations of local structure are in a surface area of the cellular form of PrP that has previously been implicated in intermolecular interactions related both to the species barrier for infectious transmission of prion disease and to immune reactions. P rion proteins (PrP) are associated with transmissible spongiform encephalopathies (TSE), which are invariably fatal diseases characterized by loss of motor control, dementia, and paralysis wasting (1, 2). Human TSEs include Creutzfeldt-Jakob disease, fatal familial insomnia, the Gerstmann-Sträussler-Scheinker syndrome, and kuru, and there is bovine spongiform encephalopathy in cattle and scrapie in sheep. The ''proteinonly'' hypothesis (3, 4) proposes that TSEs are caused by the conversion of a ubiquitous ''cellular form'' of PrP (PrP C ) into an aggregated ''scrapie form'' (PrP Sc ). According to this model, the prion protein (PrP) would at the same time be target and infectious agent in TSEs, which could explain that this class of diseases can be traced to infectious, inherited, and spontaneous origins (2, 5). PrP Sc is characterized by a high ␤-sheet content, insolubility in detergents, and resistance to proteolysis in its aggregated form (6-8) whereas PrP C is a soluble protein with a high content of ␣-helices (8, 9) and high susceptibility to proteolytic digestion. No chemical modifications have as yet been identified by which the two PrP forms would differ (10).Considering that the protein-only hypothesis suggests a change of protein conformation as a possible cause of the onset of TSEs, the three-dimensional prion protein structures have attracted keen interest. So far, nuclear magnetic resonance (NMR) solution studies have been described for monomeric, cellular forms of PrP of the two most widely used laboratory animals in prion research, the mouse (m) and the Syrian hamster (sh)...

Highlights d Structures of FUS ZnF bound to GGU and FUS RRM bound to an RNA stem loop were solved d The RGG motif destabilizes the RNA structure, suggesting a role in remodeling RNA d The RGG motif increases RNA binding without forming an ordered structure d RNA binding by the ZnF and RRM domains contribute to FUSmediated splicing functions

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...

An efficient method is presented for the production of intact mammalian prion proteins and partial sequences thereof. As an illustration we describe the production of polypeptides comprising residues 23-231, 81-231, 90-231 and 121-231 of the human prion protein (APrP) 1 . Polypeptides were expressed as histidine tail fusion proteins into inclusion bodies in the cytoplasm of Escherichia coli and refolded and oxidized while N-terminally immobilized on a nickel-NTA agarose resin. This 'high-affinity column refolding' facilitates the preparation of prion proteins by preventing protein aggregation and intermolecular disulfide formation. After elution from the resin the histidine tail can be removed using thrombin without cleaving the prion protein polypeptide chain. The same protocol as used here for APrP has been successfully applied with bovine and murine prion proteins. The protein preparations are stable for weeks at room temperature in concentrated solution and are thus suitable for detailed structural studies. Preliminary biophysical characterization of APrP(23-231) suggests that the C-terminal half of the polypeptide chain forms a well-structured globular domain, and that the N-terminal half does not form extensive regular secondary structures.

Ribonucleoproteins (RNPs) are key regulators of cellular function. We established an efficient approach that combines segmental isotope labeling of RNA with photo-crosslinking and tandem mass spectrometry to localize protein-RNA interactions simultaneously at amino acid and nucleotide resolution. The approach was tested on Polypyrimidine Tract Binding Protein 1 and U1 small nuclear RNP and the results support integrative atomic-scale structural modeling thus providing mechanistic insights into RNP regulated processes.