Probing copper2+ binding to the prion protein using diamagnetic nickel2+ and 1H NMR: the unstructured N terminus facilitates the coordination of six copper2+ ions at physiological concentrations

The prion protein (PrP) is a Cu2+ binding cell surface glyco-protein. Misfolding of PrP into a beta-sheet rich conformation is associated with transmissible spongiform encephalopathies. Here we use Ni2+ as a diamagnetic probe to further understand Cu2+ binding to PrP. Like Cu2+, Ni2+ preferentially binds to an unstructured region between residues 90 and 126 of PrP, which is a key region for amyloidogenicity and prion propagation. Using both 1H NMR and visible-circular dichroism (CD) spectroscopy, we show that two Ni2+ ions bind to His96 and His111 independently of each other. 1H NMR indicates that both Ni2+ binding sites form square-planar diamagnetic complexes. We have previously shown that Cu2+ forms a paramagnetic square-planar complex in this region, suggesting that Ni2+ could be used as a probe for Cu2+ binding. In addition, competition studies show that two Cu2+ ions can displace Ni2+ from these sites. Upon Ni2+ addition 1H NMR changes in chemical shifts indicate the imidazole ring and amide nitrogen atoms to the N terminus of both His96 and His111 act as coordinating ligands. Use of peptide fragments confirm that PrP(92-96) and PrP(107-111) represent the minimal binding motif for the two Ni2+ binding sites. Analysis of Cu2+ loaded visible-CD spectra show that as with Ni2+, PrP(90-115) binds two Cu2+ ions at His96 and His111 independently of each other. Visible CD studies with PrP(23-231Delta51-90), a construct of PrP(23-231) with the octarepeat region deleted to improve solubility, confirm binding of Ni2+ to His96 and His111 in octarepeat deleted PrP(23-231). The structure of the Cu/Ni complexes is discussed in terms of the implications for prion protein function and disease.     

The role of the 132-160 region in prion protein conformational transitions

The native conformation of host-encoded cellular prion protein (PrP(C)) is metastable. As a result of a post-translational event, PrP(C) can convert to the scrapie form (PrP(Sc)), which emerges as the essential constituent of infectious prions. Despite thorough research, the mechanism underlying this conformational transition remains unknown. However, several studies have highlighted the importance of the N-terminal region spanning residues 90-154 in PrP folding. In order to understand why PrP folds into two different conformational states exhibiting distinct secondary and tertiary structure, and to gain insight into the involvement of this particular region in PrP transconformation, we studied the pressure-induced unfolding/ refolding of recombinant Syrian hamster PrP expanding from residues 90-231, and compared it with heat unfolding. By using two intrinsic fluorescent variants of this protein (Y150W and F141W), conformational changes confined to the 132-160 segment were monitored. Multiple conformational states of the Trp variants, characterized by their spectroscopic properties (fluorescence and UV absorbance in the fourth derivative mode), were achieved by tuning the experimental conditions of pressure and temperature. Further insight into unexplored conformational states of the prion protein, likely to mimic the in vivo structural change, was obtained from pressure-assisted cold unfolding. Furthermore, salt-induced conformational changes suggested a structural stabilizing role of Tyr150 and Phe141 residues, slowing down the conversion to a beta-sheet form.     

Aggregation and fibrillization of the recombinant human prion protein huPrP90-231

According to the "protein-only" hypothesis, the critical step in the pathogenesis of prion diseases is the conformational transition between the normal (PrP(C)) and pathological (PrP(Sc)) isoforms of prion protein. To gain insight into the mechanism of this transition, we have characterized the biophysical properties of the recombinant protein corresponding to residues 90-231 of the human prion protein (huPrP90-231). Incubation of the protein under acidic conditions (pH 3.6-5) in the presence of 1 M guanidine-HCl resulted in a time-dependent transition from an alpha-helical conformation to a beta-sheet structure and oligomerization of huPrP90-231 into large molecular weight aggregates. No stable monomeric beta-sheet-rich folding intermediate of the protein could be detected in the present experiments. Kinetic analysis of the data indicates that the formation of beta-sheet structure and protein oligomerization likely occur concomitantly. The beta-sheet-rich oligomers were characterized by a markedly increased resistance to proteinase K digestion and a fibrillar morphology (i.e., they had the essential physicochemical properties of PrP(Sc)). Contrary to previous suggestions, the conversion of the recombinant prion protein into a PrP(Sc)-like form could be accomplished under nonreducing conditions, without the need to disrupt the disulfide bond. Experiments in urea indicate that, in addition to acidic pH, another critical factor controlling the transition of huPrP90-231 to an oligomeric beta-sheet structure is the presence of salt.     

Lipid interaction converts prion protein to a PrPSc-like proteinase K-resistant conformation under physiological conditions

The conversion of prion protein (PrP) to the pathogenic PrPSc conformation is central to prion disease. Previous studies revealed that PrP interacts with lipids and the interaction induces PrP conformational changes, yet it remains unclear whether in the absence of any denaturing treatment, PrP-lipid interaction is sufficient to convert PrP to the classic proteinase K-resistant conformation. Using recombinant mouse PrP, we analyzed PrP-lipid interaction under physiological conditions and followed lipid-induced PrP conformational change with proteinase K (PK) digestion. We found that the PrP-lipid interaction was initiated by electrostatic contact and followed by hydrophobic interaction. The PrP-lipid interaction converted full-length alpha-helix-rich recombinant PrP to different forms. A significant portion of PrP gained a conformation reminiscent of PrPSc, with a PrPSc-like PK-resistant core and increased beta-sheet content. The efficiency for lipid-induced PrP conversion depended on lipid headgroup structure and/or the arrangement of lipids on the surface of vesicles. When lipid vesicles were disrupted by Triton X-100, PrP aggregation was necessary to maintain the lipid-induced PrPSc-like conformation. However, the PK resistance of lipid-induced PrPSc-like conformation does not depend on amyloid fiber formation. Our results clearly revealed that the lipid interaction can overcome the energy barrier and convert full-length alpha-helix-rich PrP to a PrPSc-like conformation under physiological conditions, supporting the relevance of lipid-induced PrP conformational change to in vivo PrP conversion.     

Concealment of epitope by reduction and alkylation in prion protein

Conversion of the cellular prion protein (PrP(C)) into its pathological isoform (PrP(Sc)), the key molecular event in the pathogenesis of prion diseases, is accompanied by a conformational transition of alpha-helix into beta-sheet structures involving alpha-helix 1 (alpha1) domain from residues 144 to 154 of the protein. Reduction and alkylation of PrP(C) have been found to inhibit the conversion of PrP(C) into PrP(Sc) in vitro. Here we report that while antibody affinity of epitopes in the N- and C-terminal domains remained unchanged, reduction and alkylation of the PrP molecule induced complete concealment of an epitope in alpha1 for anti-PrP antibody 6H4 that is able to cure prion infection in the cell model. Mass spectrometric analysis of recombinant PrP showed that the alkylation reaction takes place at reduced cysteines but no modification was observed in this cryptic epitope. Our study suggests that reduction and alkylation result in local or global rearrangement of PrP tertiary structure that is maintained in both liquid and solid phases. The implications in the conversion of PrP(C) into PrP(Sc) and the therapeutics of prion diseases are discussed.     

Preferential Cu2+ coordination by His96 and His111 induces beta-sheet formation in the unstructured amyloidogenic region of the prion protein

The prion protein (PrP) is a Cu(2+) binding cell surface glycoprotein that can misfold into a beta-sheet-rich conformation to cause prion diseases. The majority of copper binding studies have concentrated on the octarepeat region of PrP. However, using a range of spectroscopic techniques, we show that copper binds preferentially to an unstructured region of PrP between residues 90 and 115, outside of the octarepeat domain. Comparison of recombinant PrP with PrP-(91-115) indicates that this prion fragment is a good model for Cu(2+) binding to the full-length protein. In contrast to previous reports we show that Cu(2+) binds to this region of PrP with a nanomolar dissociation constant. NMR and EPR spectroscopy indicate a square-planar or square-pyramidal Cu(2+) coordination utilizing histidine residues. Studies with PrP analogues show that the high affinity site requires both His(96) and His(111) as Cu(2+) ligands, rather than a complex centered on His(96) as has been previously suggested. Our circular dichroism studies indicate a loss of irregular structure on copper coordination with an increase in beta-sheet conformation. It has been shown that this unstructured region, between residues 90 and 120, is vital for prion propagation and different strains of prion disease have been linked with copper binding. The role of Cu(2+) in prion misfolding and disease must now be re-evaluated in the light of these findings.     

Characterization of 2'-fluoro-RNA aptamers that bind preferentially to disease-associated conformations of prion protein and inhibit conversion

We have isolated artificial ligands or aptamers for infectious prions in order to investigate conformational aspects of prion pathogenesis. The aptamers are 2'-fluoro-modified RNA produced by in vitro selection from a large, randomized library. One of these ligands (aptamer SAF-93) had more than 10-fold higher affinity for PrPSc than for recombinant PrPC and inhibited the accumulation of PrPres in near physiological cell-free conversion assay. To understand the molecular basis of these properties and to distinguish specific from non-specific aptamer-PrP interactions, we studied deletion mutants of bovine PrP in denatured, alpha-helix-rich and beta-sheet-rich forms. We provide evidence that, like scrapie-associated fibrils (SAF), the beta-oligomer of PrP bound to SAF-93 with at least 10-fold higher affinity than did the alpha-form. This differential affinity could be explained by the existence of two binding sites within the PrP molecule. Site 1 lies within residues 23-110 in the unstructured N terminus and is a nonspecific RNA binding site found in all forms of PrP. The region between residue 90 and 110 forms a hinge region that is occluded in the alpha-rich form of PrP but becomes exposed in the denatured form of PrP. Site 2 lies in the region C-terminal of residue 110. This site is beta-sheet conformation-specific and is not recognized by control RNAs. Taken together, these data provide for the first time a specific ligand for a disease conformation-associated site in a region of PrP critical for conformational conversion. This aptamer could provide tools for the further analysis of the processes of PrP misfolding during prion disease and leads for the development of diagnostic and therapeutic approaches to TSEs.     

Disease-associated F198S mutation increases the propensity of the recombinant prion protein for conformational conversion to scrapie-like form

The critical step in the pathogenesis of transmissible spongiform encephalopathies (prion diseases) is the conversion of a cellular prion protein (PrP(c)) into a protease-resistant, beta-sheet rich form (PrP(Sc)). Although the disease transmission normally requires direct interaction between exogenous PrP(Sc) and endogenous PrP(C), the pathogenic process in hereditary prion diseases appears to develop spontaneously (i.e. not requiring infection with exogenous PrP(Sc)). To gain insight into the molecular basis of hereditary spongiform encephalopathies, we have characterized the biophysical properties of the recombinant human prion protein variant containing the mutation (Phe(198) --> Ser) associated with familial Gerstmann-Straussler-Scheinker disease. Compared with the wild-type protein, the F198S variant shows a dramatically increased propensity to self-associate into beta-sheet-rich oligomers. In a guanidine HCl-containing buffer, the transition of the F198S variant from a normal alpha-helical conformation into an oligomeric beta-sheet structure is about 50 times faster than that of the wild-type protein. Importantly, in contrast to the wild-type PrP, the mutant protein undergoes a spontaneous conversion to oligomeric beta-sheet structure even in the absence of guanidine HCl or any other denaturants. In addition to beta-sheet structure, the oligomeric form of the protein is characterized by partial resistance to proteinase K digestion, affinity for amyloid-specific dye, thioflavine T, and fibrillar morphology. The increased propensity of the F198S variant to undergo a conversion to a PrP(Sc)-like form correlates with a markedly decreased thermodynamic stability of the native alpha-helical conformer of the mutant protein. This correlation supports the notion that partially unfolded intermediates may be involved in conformational conversion of the prion protein.     

Exploring the propensities of helices in PrP(C) to form beta sheet using NMR structures and sequence alignments

Neurodegenerative diseases induced by transmissible spongiform encephalopathies are associated with prions. The most spectacular event in the formation of the infectious scrapie form, referred to as PrP(Sc), is the conformational change from the predominantly alpha-helical conformation of PrP(C) to the PrP(Sc) state that is rich in beta-sheet content. Using sequence alignments and structural analysis of the available nuclear magnetic resonance structures of PrP(C), we explore the propensities of helices in PrP(C) to be in a beta-strand conformation. Comparison of a number of structural characteristics (such as solvent accessible area, distribution of (Phi, Psi) angles, mismatches in hydrogen bonds, nature of residues in local and nonlocal contacts, distribution of regular densities of amino acids, clustering of hydrophobic and hydrophilic residues in helices) between PrP(C) structures and a databank of "normal" proteins shows that the most unusual features are found in helix 2 (H2) (residues 172-194) followed by helix 1 (H1) (residues 144-153). In particular, the C-terminal residues in H2 are frustrated in their helical state. The databank of normal proteins consists of 58 helical proteins, 36 alpha+beta proteins, and 31 beta-sheet proteins. Our conclusions are also substantiated by gapless threading calculations that show that the normalized Z-scores of prion proteins are similar to those of other alpha+beta proteins with low helical content. Application of the recently introduced notion of discordance, namely, incompatibility of the predicted and observed secondary structures, also points to the frustration of H2 not only in the wild type but also in mutants of human PrP(C). This suggests that the instability of PrP(C) proteins may play a role in their being susceptible to the profound conformational change. Our analysis shows that, in addition to the previously proposed role for the segment (90-120) and possibly H1, the C-terminus of H2 and possibly N-terminus may play a role in the alpha-->beta transition. An implication of our results is that the ease of polymerization depends on the unfolding rate of the monomer. Sequence alignments show that helices in avian prion proteins (chicken, duck, crane) are better accommodated in a helical state, which might explain the absence of PrP(Sc) formation over finite time scales in these species. From this analysis, we predict that correlated mutations that reduce the frustration in the second half of helix 2 in mammalian prion proteins could inhibit the formation of PrP(Sc).     

Characterization of beta-sheet structure in Ure2p1-89 yeast prion fibrils by solid-state nuclear magnetic resonance

Residues 1-89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state nuclear magnetic resonance (NMR) measurements that probe the molecular structure of amyloid fibrils formed by Ure2p1-89 in vitro. Data include measurements of intermolecular magnetic dipole-dipole couplings in samples that are 13C-labeled at specific sites and two-dimensional 15N-13C and 13C-13C NMR spectra of samples that are uniformly 15N- and 13C-labeled. Intermolecular dipole-dipole couplings indicate that the beta-sheets in Ure2p1-89 fibrils have an in-register parallel structure. An in-register parallel beta-sheet structure permits polar zipper interactions among side chains of Gln and Asn residues and explains the tolerance of [URE3] to scrambling of the sequence in residues 1-89. Two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils, even when fully hydrated, show NMR linewidths that exceed those in solid-state NMR spectra of fibrils formed by residues 218-289 of the HET-s prion protein of Podospora anserina [as originally reported in Siemer, A. B., Ritter, C., Ernst, M., Riek, R., and Meier, B. H. (2005) Angew. Chem., Int. Ed. 44, 2441-2444 and confirmed by measurements reported here] by factors of three or more, indicating a lower degree of structural order at the molecular level in Ure2p1-89 fibrils. The very high degree of structural order in HET-s fibrils indicated by solid-state NMR data is therefore not a universal characteristic of prion proteins, and is likely to be a consequence of the evolved biological function of HET-s in heterokaryon incompatibility. Analysis of cross peak intensities in two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils suggests that certain portions of the amino acid sequence may not participate in a rigid beta-sheet structure, possibly including portions of the Asn-rich segment between residues 44 and 76.     

The crystal structure of the globular domain of sheep prion protein

The prion protein PrP is a naturally occurring polypeptide that becomes transformed from a normal conformation to that of an aggregated form, characteristic of pathological states in fatal transmissible spongiform conditions such as Creutzfeld-Jacob Disease and Bovine Spongiform Encephalopathy. We report the crystal structure, at 2 A resolution, of residues 123-230 of the C-terminal globular domain of the ARQ allele of sheep prion protein (PrP). The asymmetric unit contains a single molecule whose secondary structure and overall organisation correspond to those structures of PrPs from various mammalian species determined by NMR. The globular domain shows a close association of helix-1, the C-terminal portion of helix-2 and the N-terminal portion of helix-3, bounded by the intramolecular disulphide bond, 179-214. The loop 164-177, between beta2 and helix-2 is relatively well structured compared to the human PrP NMR structure. Analysis of the sheep PrP structure identifies two possible loci for the initiation of beta-sheet mediated polymerisation. One of these comprises the beta-strand, residues 129-131 that forms an intra-molecular beta-sheet with residues 161-163. This strand is involved in lattice contacts about a crystal dyad to generate a four-stranded intermolecular beta-sheet between neighbouring molecules. The second locus involves the region 188-204, which modelling suggests is able to undergo a partial alpha-->beta switch within the monomer. These loci provide sites within the PrPc monomer that could readily give rise to early intermediate species on the pathway to the formation of aggregated PrPSc containing additional intermolecular beta-structure.     

Clustered negative charges on the lipid membrane surface induce beta-sheet formation of prion protein fragment 106-126

The conformational conversion of prion protein (PrP) from an alpha-helix-rich normal cellular isoform (PrPC) to a beta-sheet-rich pathogenic isoform (PrPSc) is a key event in the development of prion diseases, and it takes place in caveolae, cavelike invaginations of the plasma membrane. A peptide homologous to residues 106-126 of human PrP (PrP106-126) is known to share several properties with PrPSc, e.g., the capability to form a beta-sheet and toxicity against PrPC-expressing cells. PrP106-126 is thus expected to represent a segment of PrP that is involved in the formation of PrPSc. We have examined the effect of lipid membranes containing negatively charged ganglioside, an important component of caveolae, on the secondary structure of PrP106-126 by circular dichroism. The peptide forms an alpha-helical or a beta-sheet structure on the ganglioside-containing membranes. The beta-sheet content increases with an increase of the peptide:lipid ratio, indicating that the beta-sheet formation is linked with self-association of the positively charged peptide on the negatively charged membrane surface. Analogous beta-sheet formation is also induced by membranes composed of negatively charged and neutral glycerophospholipids with high and low melting temperatures, respectively, in which lateral phase separation and clustering of negatively charged lipids occur as shown by Raman spectroscopy. Since ganglioside-containing membranes also exhibit lateral phase separation, clustered negative charges are concluded to be responsible for the beta-sheet formation of PrP106-126. In caveolae, clustered ganglioside molecules are likely to interact with the residue 106-126 region of PrPC to promote the PrPC-to-PrPSc conversion.     

The Val-210-Ile pathogenic Creutzfeldt-Jakob disease mutation increases both the helical and aggregation propensities of a sequence corresponding to helix-3 of PrP(C)

A peptide corresponding to the third helical region within the PrP(C) protein, from residues 198 to 218 (helix-3), was synthesised with and without the familial 210-Val to Ile Creutzfeldt-Jakob disease mutation. The NMR structure of PrP(C) predicts no global variation in stability for this mutation, indicating that local sequence rather than global structural factors are involved in the pathological effects of this mutation. 1H NMR analysis of peptides with and without this mutation indicated that it had no significant effect on local helical structure. Temperature denaturation studies monitored by CD showed that the mutation increased the helical content within this region (helical propensity), but did not stabilise the helix toward denaturation (helical stability). Aggregation data indicated that, in addition to increasing helical propensity, this mutation increased the aggregation propensity of this sequence. CD and NMR data indicate that helical interactions, stabilised by the Val-210-Ile mutation, may precede the formation of beta-sheet aggregates in this peptide sequence. Therefore, this pathological mutation probably does not facilitate PrP(C) to PrP(Sc) conversion by directly destabilising the helical structure of PrP(C), but may preferentially stabilise PrP(Sc) by facilitating beta-sheet formation within this sequence region of PrP. In addition, helical interactions between helix-3 in two or more PrP(C) molecules may promote conversion to PrP(Sc).     

Definable equilibrium states in the folding of human prion protein

The role of conformational intermediates in the conversion of prion protein from its normal cellular form (PrP(C)) to the disease-associated "scrapie" form (PrP(Sc)) remains unknown. To look for such intermediates in equilibrium conditions, we have examined the unfolding transitions of PrP(C), primarily using the chemical denaturant guanidine hydrochloride (GuHCl). When the protein conformation is assessed by NMR, there is a gradual shift of NMR signals in the regions between residues 125-146 and 186-196. The denaturant dependence of these shifts shows that in aqueous solution the native and locally unfolded conformations are both significantly populated. Following this shift, there is the major unfolding transition to generate a substantially unfolded population. However, analysis of NMR chemical shift and intensity changes shows that there is persistent structure in the molecule well beyond this major cooperative unfolding transition. Residual structure within this state is extensive and encompasses the majority of the secondary structure elements found in the native state of the protein.     

Flexible N-terminal region of prion protein influences conformation of protease-resistant prion protein isoforms associated with cross-species scrapie infection in vivo and in vitro

Transmissible spongiform encephalopathy (TSE) diseases are characterized by the accumulation in brain of an abnormal protease-resistant form of the host-encoded prion protein (PrP), PrP-res. PrP-res conformation differs among TSE agents derived from various sources, and these conformational differences are thought to influence the biological characteristics of these agents. In this study, we introduced deletions into the flexible N-terminal region of PrP (residues 34-124) and investigated the effect of this region on the conformation of PrP-res generated in an in vitro cell-free conversion assay. PrP deleted from residues 34 to 99 generated 12-16-kDa protease-resistant bands with intact C termini but variable N termini. The variable N termini were the result of exposure of new protease cleavage sites in PrP-res between residues 130 and 157, suggesting that these new cleavage sites were caused by alterations in the conformation of the PrP-res generated. Similarly truncated 12-16-kDa PrP bands were also identified in brain homogenates from mice infected with mouse-passaged hamster scrapie as well as in the cell-free conversion assay using conditions that mimicked the hamster/mouse species barrier to infection. Thus, by its effects on PrP-res conformation, the flexible N-terminal region of PrP seemed to influence TSE pathogenesis and cross-species TSE transmission.     

Local structural plasticity of the prion protein. Analysis of NMR relaxation dynamics

A template-assisted conformational change of the cellular prion protein (PrP(C)) from a predominantly helical structure to an amyloid-type structure with a higher proportion of beta-sheet is thought to be the causative factor in prion diseases. Since flexibility of the polypeptide is likely to contribute to the ability of PrP(C) to undergo the conformational change that leads to the infective state, we have undertaken a comprehensive examination of the dynamics of two recombinant Syrian hamster PrP fragments, PrP(29-231) and PrP(90-231), using (15)N NMR relaxation measurements. The molecular motions of these PrP fragments have been studied in solution using (15)N longitudinal (T(1)) and transverse relaxation (T(2)) measurements as well as [(1)H]-(15)N nuclear Overhauser effects (NOE). These data have been analyzed using both reduced spectral density mapping and the Lipari-Szabo model free formalism. The relaxation properties of the common regions of PrP(29-231) and PrP(90-231) are very similar; both have a relatively inflexible globular domain (residues 128-227) with a highly flexible and largely unstructured N-terminal domain. Residues 29-89 of PrP(29-231), which include the copper-binding octarepeat sequences, are also highly flexible. Analysis of the spectral densities at each residue indicates that even within the structured core of PrP(C), a markedly diverse range of motions is observed, consistent with the inherent plasticity of the protein. The central portions of helices B and C form a relatively rigid core, which is stabilized by the presence of an interhelix disulfide bond. Of the remainder of the globular domain, the parts that are not in direct contact with the rigid region, including helix A, are more flexible. Most significantly, slow conformational fluctuations on a millisecond to microsecond time scale are observed for the small beta-sheet. These results are consistent with the hypothesis that the infectious, scrapie form of the protein PrP(Sc) could contain a helical core consisting of helices B and C, similar in structure to the cellular form PrP(C). Our results indicate that residues 90-140, which are required for prion infectivity, are relatively flexible in PrP(C), consistent with a lowered thermodynamic barrier to a template-assisted conformational change to the infectious beta-sheet-rich scrapie isoform.     

Determination of solution conformations of PrP106-126, a neurotoxic fragment of prion protein, by 1H NMR and restrained molecular dynamics.

Experimental two-dimensional 1H NMR data have been obtained for PrP106-128 under the following solvent conditions: deionized water/2, 2,2-trifluoroethanol 50 : 50 (v/v) and dimethylsulfoxide. These data were analyzed by restrained molecular mechanics calculations to determine how changes in solvation affect the conformation of the peptide. In deionized water at pH 3.5, the peptide adopted a helical conformation in the hydrophobic region spanning residues Met112-Leu125, with the most populated helical region corresponding to the Ala115-Ala119 segment ( approximately 10%). In trifluoroethanol/H2O, the alpha-helix increased in population especially in the Gly119-Val122 tract ( approximately 25%). The conformation of this region was found to be remarkably sensitive to pH, as the Ala120-Gly124 tract shifted to an extended conformation at pH 7. In dimethylsulfoxide, the hydrophobic cluster adopted a prevalently extended conformation. For all tested solvents the region spanning residues Asn108-Met112 was present in a 'turn-like' conformation and included His111, situated just before the starting point of the alpha-helix. Rather than by conformational changes, the effect of His111 is exerted by changes in its hydrophobicity, triggering aggregation. The amphiphilic properties and the pH-dependent ionizable side-chain of His111 may thus be important for the modulation of the conformational mobility and heterogeneity of PrP106-126.     

Structural polymorphism in amyloids: new insights from studies with Y145Stop prion protein fibrils

The C-terminally-truncated human prion protein variant Y145Stop (or PrP23-144), associated with a familial prion disease, provides a valuable model for studying the fundamental properties of protein amyloids. In previous solid-state NMR experiments, we established that the β-sheet core of the PrP23-144 amyloid is composed of two β-strand regions encompassing residues ～113-125 and ～130-140. The former segment contains a highly conserved hydrophobic palindrome sequence, (113)AGAAAAGA(120), which has been considered essential to PrP conformational conversion. Here, we examine the role of this segment in fibrillization of PrP23-144 using a deletion variant, Δ113-120 PrP23-144, in which the palindrome sequence is missing. Surprisingly, we find that deletion of the palindrome sequence affects neither the amyloidogenicity nor the polymerization kinetics of PrP23-144, although it does alter amyloid conformation and morphology. Using two-dimensional and three-dimensional solid-state NMR methods, we find that Δ113-120 PrP23-144 fibrils contain an altered β-core extended N-terminally to residue ～106, encompassing residues not present in the core of wild-type PrP23-144 fibrils. The C-terminal β-strand of the core, however, is similar in both fibril types. Collectively, these data indicate that amyloid cores of PrP23-144 variants contain "essential" (i.e. nucleation-determining) and "nonessential" regions, with the latter being "movable" in amino acid sequence space. These findings reveal an intriguing new mechanism for structural polymorphism in amyloids and suggest a potential means for modulating the physicochemical properties of amyloid fibrils without compromising their polymerization characteristics.     

Comparative structure analysis of tyrosine and valine residues in unprocessed silk fibroin (silk I) and in the processed silk fiber (silk II) from Bombyx mori using solid-state (13)C,(15)N, and (2)H NMR

The solid-state (13)C CP-MAS NMR spectra of biosynthetically labeled [(13)C(alpha)]Tyr, [(13)C(beta)]Tyr, and [(13)C(alpha)]Val silk fibroin samples of Bombyx mori, in silk I (the solid-state structure before spinning) and silk II (the solid-state structure after spinning) forms, have been examined to gain insight into the conformational preferences of the semicrystalline regions. To establish the relationship between the primary structure of B. mori silk fibroin and the "local" structure, the conformation-dependent (13)C chemical shift contour plots for Tyr C(alpha), Tyr C(beta), and Val C(alpha) carbons were generated from the atomic coordinates of high-resolution crystal structures of 40 proteins and their characteristic (13)C isotropic NMR chemical shifts. From comparison of the observed Tyr C(alpha) and Tyr C(beta) chemical shifts with those predicted by the contour plots, there is strong evidence in favor of an antiparallel beta-sheet structure of the Tyr residues in the silk fibroin fibers. On the other hand, Tyr residues take a random coil conformation in the fibroin film with a silk I form. The Val residues are likely to assume a structure similar to those of Tyr residues in silk fiber and film. Solid-state (2)H NMR measurements of [3,3-(2)H(2)]Tyr-labeled B. mori silk fibroin indicate that the local mobility of the backbone and the C(alpha)-C(beta) bond is essentially "static" in both silk I and silk II forms. The orientation-dependent (i.e., parallel and perpendicular to the magnetic field) solid-state (15)N NMR spectra of biosynthetically labeled [(15)N]Tyr and [(15)N]Val silk fibers reveal the presence of highly oriented semicrystalline regions.     

Crystal structure of the human prion protein reveals a mechanism for oligomerization

The pathogenesis of transmissible encephalopathies is associated with the conversion of the cellular prion protein, PrP(C), into a conformationally altered oligomeric form, PrP(Sc). Here we report the crystal structure of the human prion protein in dimer form at 2 A resolution. The dimer results from the three-dimensional swapping of the C-terminal helix 3 and rearrangement of the disulfide bond. An interchain two-stranded antiparallel beta-sheet is formed at the dimer interface by residues that are located in helix 2 in the monomeric NMR structures. Familial prion disease mutations map to the regions directly involved in helix swapping. This crystal structure suggests that oligomerization through 3D domain-swapping may constitute an important step on the pathway of the PrP(C) --> PrP(Sc) conversion.