A novel copper(II) coordination at His186 in full-length murine prion protein

To explore Cu(II) ion coordination by His¹⁸⁶ in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique). Six moPrP mutants, moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C), and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one. Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1). This line broadening indicated the presence of electron-electron dipolar interaction between Cu(II) and the nitroxide spin probe, suggesting that each interspin distance was within 20 Å. The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12.1 Å, 18.1 Å, 10.7 Å, and 8.4 Å, respectively. In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed. These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrP^C.     

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.     

Solid-state NMR studies of the prion protein H1 fragment.

Conformational changes in the prion protein (PrP) seem to be responsible for prion diseases. We have used conformation-dependent chemical-shift measurements and rotational-resonance distance measurements to analyze the conformation of solid-state peptides lacking long-range order, corresponding to a region of PrP designated H1. This region is predicted to undergo a transformation of secondary structure in generating the infectious form of the protein. Solid-state NMR spectra of specifically 13C-enriched samples of H1, residues 109-122 (MKHMAGAAAAGAVV) of Syrian hamster PrP, have been acquired under cross-polarization and magic-angle spinning conditions. Samples lyophilized from 50% acetonitrile/50% water show chemical shifts characteristic of a beta-sheet conformation in the region corresponding to residues 112-121, whereas samples lyophilized from hexafluoroisopropanol display shifts indicative of alpha-helical secondary structure in the region corresponding to residues 113-117. Complete conversion to the helical conformation was not observed and conversion from alpha-helix back to beta-sheet, as inferred from the solid-state NMR spectra, occurred when samples were exposed to water. Rotational-resonance experiments were performed on seven doubly 13C-labeled H1 samples dried from water. Measured distances suggest that the peptide is in an extended, possibly beta-strand, conformation. These results are consistent with the experimental observation that PrP can exist in different conformational states and with structural predictions based on biological data and theoretical modeling that suggest that H1 may play a key role in the conformational transition involved in the development of prion diseases.     

Dynamics and local ordering of spin-labeled prion protein: an ESR simulation study of a highly PH-sensitive site

Valine 160 on beta-sheet-2 (S2) of mouse prion (moPrPC) has been previously identified as the most highly pH-sensitive site on moPrPC by ESR spectroscopy using site-directed spin labeling (SDSL) technique. However, no further theoretical analysis to reveal the molecular dynamics reported on the experimental ESR spectra is available. The X-band ESR spectra of R1 nitroxide spin label at V160 and four other sites are carefully analyzed over large pH and temperature ranges using a spectral simulation method based upon stochastic Liouville equation (SLE). The results clearly reveal the dynamics and ordering of the local environment of V160R1 showing that (i) molecular mobility of V160R1 on S2 gradually increases with a decrease of pH from 7.5 to 4.5; (ii) two distinctly different spectral components are simultaneously present in all spectra of V160R1 studied. The spectral components are, respectively, denoted as immobile (Im), characterized by lower molecular mobility and higher ordering, and mobile (Mb) component of high mobility and low ordering. The population ratio (Im/Mb) increases with increasing pH, while Im remains dominant in all V160R1 spectra. It suggests a more mobile and disordered dynamic molecular structure for mouse PrPC, which is very likely correlated with increased beta-sheet content at low pH, as the environment changes from neutral to acidic pH. Together with the results of the SLE-based analyses on the spectra of other sites that appear pH-insensitive, we suggest that the simultaneous presence of the spectral components for V160R1 is strongly correlated with the coexistence of multiple protein conformations in local structure of PrPC over the varied pH range. It demonstrates that the combined approach of the SDSL technique and the SLE-based analysis leads to a powerful method for unraveling the complexity of protein dynamics.     

Folding and intrinsic stability of deletion variants of PrP(121-231), the folded C-terminal domain of the prion protein

Transmissible spongiform encephalopathies in mammals are believed to be caused by PrPSc, the insoluble, oligomeric isoform of the cellular prion protein PrPC. PrPC and the subunits of PrPSc have identical covalent but different tertiary structure. To address the question of whether parts of the structure of PrPC are sufficiently stable to be retained in PrPSc, we have constructed two deletion variants of the C-terminal PrPC domain, PrP(121-231), which is the only part of recombinant PrP with defined tertiary structure. One of the variants, H2-H3, comprises the last two alpha-helices of PrP(121-231) that have been proposed to be preserved in models of PrP(Sc). In the other variant, PrP(121-231)-deltaH1, the first alpha-helix of PrP(121-231) was deleted and replaced by introduction of the beta-turn dipeptide Asn-Gly between the strands of the single beta-sheet of PrP(121-231). Although both deletion constructs still show alpha-helical CD-spectra, they are more disordered and thermodynamically strongly destabilized compared to PrP(121-231), with free energies of folding close to zero. These data demonstrate that the tertiary structure context is critical for the conformation of the segment comprising alpha-helix 2 and 3 in the solution structure of recombinant PrP.     

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).     

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).     

Secondary structure extensions in Pyrococcus furiosus ferredoxin destabilize the disulfide bond relative to that in other hyperthermostable ferredoxins. Global consequences for the disulfide orientational heterogeneity.

The single cubane cluster ferredoxin (Fd) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) possesses several unique properties when compared even to Fds from other hyperthermophilic archaea or bacteria. These include an equilibrium molecular heterogeneity, a six- to seven-residue increase in size, an Asp rather than the Cys as one cluster ligand, and a readily reducible disulfide bond. NMR assignments and determination of both secondary structure and tertiary contacts remote from the paramagnetic oxidized cluster of Pf 3Fe Fd with an intact disulfide bond reported previously (Teng Q., Zhou, Z. H., Smith, E. T., Busse, S. C., Howard, J. B. Adams, M. W. W., and La Mar, G. (1994) Biochemistry 33, 6316-6328) are extended here to the 4Fe oxidized cluster WT (1H and 15N) and D14C (1H only) Fds with an intact disulfide bond and to the 4Fe oxidized WT Fd (1H and 15N) with a cleaved disulfide bond. All forms are shown to possess a long (13-member) alpha-helix, two beta-sheets (one double-, one triple-stranded), and three turns outside the cluster vicinity, each with tertiary contacts among themselves as found in other Fds. While the same secondary structural elements, with similar tertiary contacts, are found in other hyperthermostable Fds, Pf Fd has two elements, the long helix and the triple-stranded beta-sheet, that exhibit extensions and form multiple tertiary contacts. All Pf Fd forms with an intact disulfide bond exhibit a dynamic equilibrium heterogeneity which is shown to modulate a hydrogen-bonding network in the hydrophobic core that radiates from the Cys21-Cys48 disulfide bond and encompasses residues Lys36, Val24, Cys21, and Cys17 and the majority of the long helix. The heterogeneity is attributed to population of the alternate S and R chiralities of the disulfide bond, each destabilized by steric interactions with the extended alpha-helix. Comparison of the chemical shifts and their temperature gradients reveals that the molecular structure of the protein with the less stable R disulfide resembles that of the Fd with a cleaved disulfide bond. Both cluster architecture (3Fe vs 4Fe) and ligand mutation (Cys for Asp14) leave the disulfide orientational heterogeneity largely unperturbed. It is concluded that the six- to seven-residue extension that results in a longer helix and larger beta-sheet in Pf Fd, relative to other hyperthermostable Fds, more likely serves to destabilize the disulfide bond, and hence make it more readily reducible, than to significantly increase protein thermostability.     

Secondary structure changes stabilize the reactive-centre cleaved form of SERPINs. A study by 1H nuclear magnetic resonance and Fourier transform infrared spectroscopy.

Proteinase inhibitor members of the SERPIN superfamily are characterized by the presence of a proteolytically sensitive reactive-site loop. Cleavage within this region results in a conformational transition from an unstable "stressed" native protein to a more stable "relaxed" cleaved molecule. In order to identify the principal molecular aspects of this transition, 1H nuclear magnetic resonance (n.m.r.) and FT-IR spectroscopy were applied to the study of four SERPINs. 1H n.m.r. spectra of approximately 20 high-field ring-current-shifted methyl signals exhibited slightly different chemical shifts in the native and cleaved forms of alpha 1-antitrypsin (alpha 1-AT), alpha 1-antichymotrypsin (alpha 1-ACT) and C1 inhibitor (C1-INH), but not ovalbumin, between 20 degrees C and 90 degrees C. Ring current calculations based on crystal co-ordinates for cleaved alpha 1-AT and alpha 1-ACT and native ovalbumin showed that these signals originate from highly localized interactions between different buried residues corresponding to alpha-helix and beta-sheet segments of the SERPIN fold. The small shift changes correspond to small relative conformational side-chain rearrangements of about 0.01 nm to 0.05 nm in the protein hydrophobic core, i.e. the tertiary structure interactions in the two forms of the SERPIN fold are well-preserved, and changes in this appear unimportant for the stabilization found after reactive centre cleavage. Fourier transform infrared (FT-IR) spectroscopic studies of the amide I band showed that the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH contain 28% to 36% alpha-helix and 38% to 44% beta-sheet. Second derivative FT-IR spectra using H2O and 2H2O buffers revealed very large differences in the amide I band between the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH, but not for ovalbumin. The alpha-helix band was most sensitive to 1H-2H exchange, while the beta-sheet bands were not, and greater amounts of antiparallel beta-sheet were detected in the cleaved form. 1H n.m.r. showed that polypeptide amide 1H-2H exchange was greater in the native forms of alpha 1-AT, alpha 1-ACT and C1-INH than in their cleaved forms, whereas for ovalbumin it was unchanged. The FT-IR and 1H-2H exchange data show that alterations in the secondary structure are central to the stabilization of the cleaved SERPIN structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solution structure of the conserved hypothetical protein Rv2302 from Mycobacterium tuberculosis

The Mycobacterium tuberculosis protein Rv2302 (80 residues; molecular mass of 8.6 kDa) has been characterized using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. While the biochemical function of Rv2302 is still unknown, recent microarray analyses show that Rv2302 is upregulated in response to starvation and overexpression of heat shock proteins and, consequently, may play a role in the biochemical processes associated with these events. Rv2302 is a monomer in solution as shown by size exclusion chromatography and NMR spectroscopy. CD spectroscopy suggests that Rv2302 partially unfolds upon heating and that this unfolding is reversible. Using NMR-based methods, the solution structure of Rv2302 was determined. The protein contains a five-strand, antiparallel beta-sheet core with one C-terminal alpha-helix (A61 to A75) nestled against its side. Hydrophobic interactions between residues in the alpha-helix and beta-strands 3 and 4 hold the alpha-helix near the beta-sheet core. The electrostatic potential on the solvent-accessible surface is primarily negative with the exception of a positive arginine pocket composed of residues R18, R70, and R74. Steady-state {(1)H}-(15)N heteronuclear nuclear Overhauser effects indicate that the protein's core is rigid on the picosecond timescale. The absence of amide cross-peaks for residues G13 to H19 in the (1)H-(15)N heteronuclear single quantum correlation spectrum suggests that this region, a loop between beta-strands 1 and 2, undergoes motion on the millisecond to microsecond timescale. Dali searches using the structure closest to the average structure do not identify any high similarities to any other known protein structure, suggesting that the structure of Rv2302 may represent a novel protein fold.     

Three-dimensional structure of the ribonuclease T1 2'-GMP complex at 1.9-A resolution

The complex formed between the enzyme ribonuclease T1 (EC 3.1.27.3) and its specific inhibitor 2'-guanylic acid (2'-GMP) has been refined to R = 0.180 using x-ray diffraction data to 1.9-A resolution. The protein molecule displays a compact fold; a 4.5 turn alpha-helix packed over an antiparallel beta-pleated sheet shields most of the hydrophobic interior of the protein against the solvent. The extended pleated sheet structure of ribonuclease T1 is composed of three long and four short strands building up a two-stranded minor beta-sheet near the amino terminus and a five-stranded major sheet in the interior of the protein molecule. In the complex with ribonuclease T1, the inhibitor 2'-guanylic acid adopts the syn-conformation and C2'-endo sugar pucker. Binding of the nucleotide is mainly achieved through amino acid residues 38-46 of the protein. The catalytically active amino acid residues of ribonuclease T1 (His40, Glu58, Arg77, and His92) are located within the major beta-sheet which, as evident from the analysis of atomic temperature factors, provides an environment of minimal local mobility. The geometry of the active site is consistent with a mechanism for phosphodiester hydrolysis where, in the transesterification step, His40 and/or Glu58 act as a general base toward the ribose 2'-hydroxyl group and His92, as a general acid, donates a proton to the leaving 5'-hydroxyl group.     

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.     

Structural characterization of the trypsin-resistant core in the nuclear sperm-specific protein from Spisula solidissima

Trypsin digestion of the protamine-like protein from Spisula solidissima has revealed the existence of an internal resistant core. The peptide contains 75 amino acid residues, and its primary structure shows some conserved sequences that are common to those found in the core of the somatic histone H5 from chicken erythrocytes. The secondary structure of this core exhibits 33% antiparallel beta-sheet, 18% beta-turns, 37% random coil, and only 10% alpha-helix, in contrast to histone H5. Hydrodynamic measurements indicate a compact globular assembly for the tertiary structure of this peptide, when compared to the more extended shape observed for the whole protein. The possible relatedness of this protein to the histone H1 family is discussed.     

Solution structure of BmP01 from the venom of scorpion Buthus martensii Karsch

From the venom of scorpion Buthus martensii Karsch,a short peptide (BmP01, 29 amino acid residues) was isolated and characterized as previously reported (Lebren, R. R., et al. (1997) Eur. J. Biochem. 245, 457-464). It was shown to reduce 33% outward K(+) channel (hippocampal neurons) currents at 10 microM. The solution structure of BmP01 was determined by 2D (1)H NMR spectroscopy. The NOEs, coupling constants, and H-D exchange obtained from NMR spectroscopy were used in structural calculations. The conformation of BmP01 is composed of a short alpha-helix (Cys 3-Thr 12) and a two-stranded antiparallel beta-sheet (Ala 15-Asp 20 and Lys 23-Pro 28). There are three disulfide bridges (Cys 3-Cys 19, Cys 6-Cys 24 and Cys 10-Cys 26) connecting the alpha-helix and beta-sheet. Asp 20 to Lys 23 form a type II turn linking the two strands. Structural and electrostatic potential comparison between BmP01 and its analogues are also presented.     

Amyloid-beta-sheet formation at the air-water interface

An amyloid(1-40) solution rich in coil, turn, and alpha-helix, but poor in beta-sheet, develops monolayers with a high beta-sheet content when spread at the air-water interface. These monolayers are resistant to repeated compression-dilatation cycles and interaction with trifluoroethanol. The secondary structure motifs were detected by circular dichroism (CD) in solution and with infrared reflection-absorption spectroscopy (IRRAS) at the interface. Hydrophobic influences are discussed for the structure conversion in an effort to understand the completely unknown reason for the natural change of the normal prion protein cellular (PrP(C)) into the abnormal prion protein scrapie (PrP(Sc)).     

Multinuclear magnetic resonance studies of the 2Fe.2S* ferredoxin from Anabaena species strain PCC 7120. 1. Sequence-specific hydrogen-1 resonance assignments and secondary structure in solution of the oxidized form

Complete sequence-specific assignments were determined for the diamagnetic 1H resonances from Anabaena 7120 ferredoxin (Mr = 11,000). A novel assignment procedure was followed whose first step was the identification of the 13C spin systems of the amino acids by a 13C(13C) double quantum correlation experiment [Oh, B.-H., Westler, M. W., Darba, P., & Markley, J. L. (1988) Science 240, 908-911]. Then, the 1H spin systems of the amino acids were identified from the 13C spin systems by means of direct and relayed 1H(13C) single-bond correlations [Oh, B.-H., Westler, W. M., & Markley, J. L. (1989) J. Am. Chem. Soc. 111, 3083-3085]. The sequential resonance assignments were based mainly on conventional interresidue 1H alpha i-1HNi + 1 NOE connectivities. Resonances from 18 residues were not resolved in two-dimensional 1H NMR spectra. When these residues were mapped onto the X-ray crystal structure of the homologous ferredoxin from Spirulina platensis [Fukuyama, K., Hase, T., Matsumoto, S., Tsukihara, T., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., & Matsubara, H. (1980) Nature 286, 522-524], it was found that they correspond to amino acids close to the paramagnetic 2Fe.2S* cluster. Cross peaks in two-dimensional homonuclear 1H NMR spectra were not observed for any protons closer than about 7.8 A to both iron atoms. Secondary structural features identified in solution include two antiparallel beta-sheets, one parallel beta-sheet, and one alpha-helix.     

Oxidation of methionine residues in the prion protein by hydrogen peroxide

Reaction of H(2)O(2) with the recombinant SHa(29-231) prion protein resulted in rapid oxidation of multiple methionine residues. Susceptibility to oxidation of individual residues, assessed by mass spectrometry after digestion with CNBr and lysC, was in general a function of solvent exposure. Met 109 and Met 112, situated in the highly flexible amino terminus, and key residues of the toxic peptide PrP (106-126), showed the greatest susceptibility. Met 129, a residue located in a polymorphic position in human PrP and modulating risk of prion disease, was also easily oxidized, as was Met 134. The structural effect of H(2)O(2)-induced methionine oxidation on PrP was studied by CD spectroscopy. As opposed to copper catalyzed oxidation, which results in extensive aggregation of PrP, this reaction led only to a modest increase in beta-sheet structure. The high number of solvent exposed methionine residues in PrP suggests their possible role as protective endogenous antioxidants.     

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 dependence of the cellular isoform of prion protein on solvent: spectroscopic characterization of an intermediate conformation.

Using circular dichroism, fluorescence, and infrared spectroscopies, we studied the secondary structure of purified hamster PrP(C) in the presence of the mild, nonionic detergent octylglucoside. Under these native conditions, PrP(C) displayed an unexpectedly high beta-sheet component, intermediate between the values previously reported for PrP(Sc) and an isoform of PrP(C) isolated in a zwitterionic detergent. The structure of PrP(C) appeared to depend strongly on the detergent and/or phase. Switching from octylglucoside to zwitterion 3-14 drastically modified PrP secondary structure by increasing the alpha-helix while abolishing the beta-sheet component. In contrast, the conformation of PrP(C) in zwitterion was highly stable, since reverting to octylglucoside did not restore the original native structure. These and other results show that native PrP(C) in octylglucoside has some of the conformational characteristics that make the protein susceptible to conversion into PrP(Sc). Most importantly, this is the first study to demonstrate the intrinsic plasticity of the full-length native PrP(C) isolated from animal brains.