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)...

The NMR structure of the globular domain of the human prion protein (hPrP) with residues 121-230 at pH 7.0 shows the same global fold as the previously published structure determined at pH 4.5. It contains three ␣-helices, comprising residues 144 -156, 174 -194, and 200 -228, and a short anti-parallel ␤-sheet, comprising residues 128 -131 and 161-164. There are slight, strictly localized, conformational changes at neutral pH when compared with acidic solution conditions: helix ␣1 is elongated at the C-terminal end with residues 153-156 forming a 3 10 -helix, and the population of helical structure in the C-terminal two turns of helix ␣2 is increased. The protonation of His 155 and His 187 presumably contributes to these structural changes. Thermal unfolding monitored by far UV CD indicates that hPrP-(121-230) is significantly more stable at neutral pH. Measurements of amide proton protection factors map local differences in protein stability within residues 154 -157 at the Cterminal end of helix ␣1 and residues 161-164 of ␤-strand 2. These two segments appear to form a separate domain that at acidic pH has a larger tendency to unfold than the overall protein structure. This domain could provide a "starting point" for pH-induced unfolding and thus may be implicated in endosomic PrP C to PrP Sc conformational transition resulting in transmissible spongiform encephalopaties.The prion protein (PrP), 1 a predominantly synaptic protein present in all higher organisms (1-4), constitutes a major component of the infectious agent (prion) that causes transmissible spongiform encephalopathies (5, 6). The normal cellular isoform of the protein, PrP C , is soluble and protease-sensitive, whereas the disease-associated ␤-sheet-rich form, PrP Sc , is insoluble, partially resistant to protease digestion (7,8), and thought to propagate by converting PrP C molecules into an alternative conformation (9 -11). Recently, it has been shown that the accumulation of even small quantities of misfolded PrP in the cytosol is strongly neurotoxic in cultured cells and transgenic mice (12, 13). However, the subcellular localization of the conformational transition of PrP C into PrP Sc is controversial (14). There are indications that it takes place either at the cell surface, where the average interstitial milieu of the brain (15, 16) has a pH of 7.3, or after internalization of PrP Sc into endosomes (17)(18)(19), where pH values range between 4.7 and 5.8 (20).The in vitro conversion of human brain PrP C to a PrP Sc -like form is enhanced at acidic pH (21). Biophysical studies have shown that the free energy of unfolding of hPrP-(90 -231) is lower at acid pH than at neutral pH (22) and that in acidic guanidinium chloride hPrP-(90 -231) forms a folding intermediate that contains a large amount of ␤-sheet secondary structure. A ␤-sheet-rich folding intermediate has also been observed for mouse PrP-(121-231) at low pH in urea but is not seen at neutral pH (23). NMR structures are available for several recombinantly expressed mammalian prion prot...

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.

A monomeric peptide fragment of GroEL, consisting of residues 191-376, is a mini-chaperone with a functional chaperoning activity. We have solved the crystal structure at 1.7 Å resolution of GroEL(191-376) with a 17-residue N-terminal tag. The N-terminal tag of one molecule binds in the active site of a neighboring molecule in the crystal. This appears to mimic the binding of a peptide substrate molecule. Seven substrate residues are bound in a relatively extended conformation. Interactions between the substrate and the active site are predominantly hydrophobic, but there are also four hydrogen bonds between the main chain of the substrate and side chains of the active site. Although the preferred conformation of a bound substrate is essentially extended, the f lexibility of the active site may allow it to accommodate the binding of exposed hydrophobic surfaces in general, such as molten globule-type structures. GroEL can therefore help unfold proteins by binding to a hydrophobic region and exert a binding pressure toward the fully unfolded state, thus acting as an ''unfoldase.'' The structure of the mini-chaperone is very similar to that of residues 191-376 in intact GroEL, so we can build it into GroEL and reconstruct how a peptide can bind to the tetradecamer. A ring of connected binding sites is noted that can explain many aspects of substrate binding and activity.