Endothelium-independent vasodilation effect of di- and tri-peptides in thoracic aorta of Sprague-Dawley rats

The goal of this study was to elucidate the structure-activity relationship for vasodilating peptides and their underlying mechanism. In this study, we synthesized 62 di- and tri-peptides having aromatic amino acid residues (Tyr, Trp and Phe). Among them, only 4 peptides (HW, WH, WL and WV) evoked an apparent vasodilating effect in 50 mM KCl-contracted aortic rings in the descending order of WH>HW>WL>WV; WH showed the vasodilating activity with an EC50 of 3.4 mM. Within our experimental results, it seems likely that Trp residue at the N-terminal would play a role in eliciting vasodilating effect. No appearance of vasodilating effect for stereoisomers of WH with D-configuration revealed that the vessel would recognize the L-configuration of WH. The presence of angiotensin I-converting enzyme (ACE) inhibitor (50 nM enalaprilat) did not affect the WH-induced vasodilating effect, though WH showed a slight ACE inhibitory activity (IC50: 93 microM). The effect was also observed in the endothelium-denuded aortic rings. In contrast, WH provoked a significant displacement to the right in the vasodilating curve in the presence of 30 nM verapamil, while no shift was observed in the presence of 2.5 nM nifedipine. These results indicate that WH, a vasodilating di-peptide, would exert a vasodilation via the suppression of Ca2+ influx into KCl-induced depolarized aortic rings. The present study also suggests that the binding site of WH to the Ca2+ channel may be competitive to that of phenylalkylamine-type voltage-gated L-type Ca2+ channel blocker.     

Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI-MS demonstrates that Cu2+ is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N-terminal domain, containing multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI-MS are pH dependent: a peptide containing four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity for peptides C-terminally extended to incorporate a fifth histidine. Dissociation constants for each Cu2+ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addition of the third and fourth Cu2+ ions to the extended peptides at pH 7.4, K(D)'s are <100 nM. N-terminal acetylation of the peptides caused some reduction in the stoichiometry of binding at both pH's. Cu2+ also binds to a peptide corresponding to the extreme N-terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu2+ complexation. Although the structure of the four-octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by circular dichroism, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu2+ transporter by binding Cu2+ ions from the extracellular medium under physiologic conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes.     

Nuclear Overhauser effects as probes of peptide structure. A diagnostic for left-handed helical conformations

The conformational dependence of the interresidue interproton distances in peptides, C alpha H ... Ni + 1 H and NiH ... Ni + 1 H, have been used to identify zones of sterically allowed phi, psi space, where both distances are less than 3A and expected to yield nuclear Overhauser effects (NOEs). L-residues in left-handed helical conformations are expected to yield both interresidue NOEs and also an appreciable intraresidue NiH----C alpha iH NOE. The effect of cutoff distances has been evaluated. Experimental results on three model peptides illustrate the utility of these NOEs in identifying L-residues at the i + 2 position of Type II and I' beta-turns. Simultaneous observation of both interresidue NOEs may also be indicative of conformational heterogeneity in specific cases, as illustrated for a single residue in a decapeptide.     

Prion disease: A loss of antioxidant function?

Prion disease, a neurodegenerative disorder, is widely believed to arise when a cellular prion protein (PrP(C)) undergoes conformational changes to a pathogenic isoform (PrP(Sc)). Recent data have shown PrP(C) to be copper binding and that it acquires antioxidant activity as a result. This enzymatic property is dependent mainly on copper binding to the octarepeats region. In normal human brain and human prion disease, there is a population of brain-derived PrP that has been truncated at the N-terminal which encompassed the octarepeats region. Increasing evidences have suggested imbalances of metal-catalyzed reactions to be the common denominator for several neurodegenerative diseases. Therefore, we propose that one of the causative factors for prion disease could be due to the imbalances in metal-catalyzed reactions resulting in an alteration of the antioxidant function. These result in an increase level of oxidative stress and, as such, trigger the neurodegenerative cascade.     

Simulation vs. reality: a comparison of in silico distance predictions with DEER and FRET measurements

Site specific incorporation of molecular probes such as fluorescent- and nitroxide spin-labels into biomolecules, and subsequent analysis by F?rster resonance energy transfer (FRET) and double electron-electron resonance (DEER) can elucidate the distance and distance-changes between the probes. However, the probes have an intrinsic conformational flexibility due to the linker by which they are conjugated to the biomolecule. This property minimizes the influence of the label side chain on the structure of the target molecule, but complicates the direct correlation of the experimental inter-label distances with the macromolecular structure or changes thereof. Simulation methods that account for the conformational flexibility and orientation of the probe(s) can be helpful in overcoming this problem. We performed distance measurements using FRET and DEER and explored different simulation techniques to predict inter-label distances using the Rpo4/7 stalk module of the M. jannaschii RNA polymerase. This is a suitable model system because it is rigid and a high-resolution X-ray structure is available. The conformations of the fluorescent labels and nitroxide spin labels on Rpo4/7 were modeled using in vacuo molecular dynamics simulations (MD) and a stochastic Monte Carlo sampling approach. For the nitroxide probes we also performed MD simulations with explicit water and carried out a rotamer library analysis. Our results show that the Monte Carlo simulations are in better agreement with experiments than the MD simulations and the rotamer library approach results in plausible distance predictions. Because the latter is the least computationally demanding of the methods we have explored, and is readily available to many researchers, it prevails as the method of choice for the interpretation of DEER distance distributions.     

Calcium-dependent structural coupling between opposing globular domains of calmodulin involves the central helix.

We have used fluorescence spectroscopy to investigate the average structure and extent of conformational heterogeneity associated with the central helix in calmodulin (CaM), a sequence that contributes to calcium binding sites 2 and 3 and connects the amino- and carboxyl-terminal globular domains. Using site-directed mutagenesis, a double mutant was constructed involving conservative substitution of Tyr(99) --> Trp(99) and Leu(69) --> Cys(69) with no significant effect on the secondary structure of CaM. These mutation sites are at opposite ends of the central helix. Trp(99) acts as a fluorescence resonance energy transfer (FRET) donor in distance measurements of the conformation of the central helix. Cys(69) provides a reactive group for the covalent attachment of the FRET acceptor 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS). AEDANS-modified CaM fully activates the plasma membrane (PM) Ca-ATPase, indicating that the native structure is retained following site-directed mutagenesis and chemical modification. We find that the average spatial separation between Trp(99) and AEDANS covalently bound to Cys(69) decreases by approximately 7 +/- 2 A upon calcium binding. However, irrespective of calcium binding, there is little change in the conformational heterogeneity associated with the central helix under physiologically relevant conditions (i.e., pH 7.5, 0.1 M KCl). These results indicate that calcium activation alters the spatial arrangement of the opposing globular domains between two defined conformations. In contrast, under conditions of low ionic strength or pH the structure of CaM is altered and the conformational heterogeneity of the central helix is decreased upon calcium activation. These results suggest the presence of important ionizable groups that affect the structure of the central helix, which may play an important role in mediating the ability of CaM to rapidly bind and activate target proteins.     

Probing the role of PrP repeats in conformational conversion and amyloid assembly of chimeric yeast prions

Oligopeptide repeats appear in many proteins that undergo conformational conversions to form amyloid, including the mammalian prion protein PrP and the yeast prion protein Sup35. Whereas the repeats in PrP have been studied more exhaustively, interpretation of these studies is confounded by the fact that many details of the PrP prion conformational conversion are not well understood. On the other hand, there is now a relatively good understanding of the factors that guide the conformational conversion of the Sup35 prion protein. To provide a general model for studying the role of oligopeptide repeats in prion conformational conversion and amyloid formation, we have substituted various numbers of the PrP octarepeats for the endogenous Sup35 repeats. The resulting chimeric proteins can adopt the [PSI+] prion state in yeast, and the stability of the prion state depends on the number of repeats. In vitro, these chimeric proteins form amyloid fibers, with more repeats leading to shorter lag phases and faster assembly rates. Both pH and the presence of metal ions modulate assembly kinetics of the chimeric proteins, and the extent of modulation is highly sensitive to the number of PrP repeats. This work offers new insight into the properties of the PrP octarepeats in amyloid assembly and prion formation. It also reveals new features of the yeast prion protein, and provides a level of control over yeast prion assembly that will be useful for future structural studies and for creating amyloid-based biomaterials.     

PrP mutants with different numbers of octarepeat sequences are more susceptible to the oxidative stress

One of the physiological functions of cellular prion protein(PrP C )is believed to work as a cellular resistance to oxidative stress,in which the octarepeats region within PrP plays an important role.However,the detailed mechanism is less clear.In this study,the expressing plasmids of wild-type PrP (PrP-PG5)and various PrP mutants containing 0(PrP-PG0),9(PrP-PG9)and 12(PrP-PG12)octarepeats were generated and PrP proteins were expressed both in E.coli and in mammalian cells.Protein aggregation and formation of carbonyl groups were clearly seen in the recombinant PrPs expressed from E.coli after treatment of H2O2.MTT and trypan blue staining assays revealed that the cells expressing the mutated PrPs within octarepeats are less viable than the cells expressing wild-type PrP.Statistically significant high levels of intracellular free radicals and low levels of glutathione peroxidase were observed in the cells transfected with plasmids containing deleted or inserted octarepeats.Remarkably more productions of carbonyl groups were detected in the cells expressing PrPs with deleted and inserted octarepeats after exposing to H2O2.Furthermore,cells expressing wild-type PrP showed stronger resistant activity to the challenge of H2O2 at certain extent than the mutated PrPs and mock. These data provided the evidences that the octarepeats number within PrP is critical for maintaining its activity of antioxidation.Loss of its protective function against oxidative stress may be one of the possible pathways for the mutated PrPs to involve in the pathogenesis of familial Creutzfeldt-Jacob diseases.     

Phosphorylation-induced conformational changes in a mitogen-activated protein kinase substrate. Implications for tyrosine hydroxylase activation

Mitogen-activated protein (MAP) kinase-mediated phosphorylation of specific residues in tyrosine hydroxylase leads to an increase in enzyme activity. However, the mechanism whereby phosphorylation affects enzyme turnover is not well understood. We used a combination of fluorescence resonance energy transfer (FRET) measurements and molecular dynamics simulations to explore the conformational free energy landscape of a 10-residue MAP kinase substrate found near the N terminus of the enzyme. This region is believed to be part of an autoregulatory sequence that overlies the active site of the enzyme. FRET was used to measure the effect of phosphorylation on the ensemble of peptide conformations, and molecular dynamics simulations generated free energy profiles for both the unphosphorylated and phosphorylated peptides. We demonstrate how FRET transfer efficiencies can be calculated from molecular dynamics simulations. For both the unphosphorylated and phosphorylated peptides, the calculated FRET efficiencies are in excellent agreement with the experimentally determined values. Moreover, the FRET measurements and molecular simulations suggest that phosphorylation causes the peptide backbone to change direction and fold into a compact structure relative to the unphosphorylated state. These results are consistent with a model of enzyme activation where phosphorylation of the MAP kinase substrate causes the N-terminal region to adopt a compact structure away from the active site. The methods we employ provide a general framework for analyzing the accessible conformational states of peptides and small molecules. Therefore, they are expected to be applicable to a variety of different systems.     

Non-natural amino acid fluorophores for one- and two-step fluorescence resonance energy transfer applications

Fluorescence resonance energy transfer (FRET) provides a powerful means to study protein conformational changes. However, the incorporation of an exogenous FRET pair into a protein could lead to undesirable structural perturbations of the native fold. One of the viable strategies to minimizing such perturbations is to use non-natural amino acid-based FRET pairs. Previously, we showed that p-cyanophenylalanine (Phe_CN) and tryptophan (Trp) constitute such a FRET pair, useful for monitoring protein folding-unfolding transitions. Here we further show that 7-azatryptophan (7AW) and 5-hydroxytryptophan (5HW) can also serve as a FRET acceptor to Phe_CN, and the resultant FRET pairs offer certain advantages over Phe_CN-Trp. For example, the fluorescence spectrum of 7AW is sufficiently separated from that of Phe_CN, making it straightforward to decompose the FRET spectrum into donor and acceptor contributions. Moreover, we show that Phe_CN, Trp, and 7AW can be used together to form a multi-FRET system, allowing more structural information to be extracted from a single FRET experiment. The applicability of these FRET systems is demonstrated in a series of studies where they are employed to monitor the urea-induced unfolding transitions of the villin headpiece subdomain (HP35), a designed ββα motif (BBA5), and the human Pin1 WW domain.     

Reversible scaling of dihedral angle barriers during molecular dynamics to improve structure prediction of cyclic peptides.

Peptide cyclization or chemical cross-linking has frequently been used to restrict the conformational freedom of a peptide, for example, to enhance its capacity for selective binding to a target receptor molecule. Structure prediction of cyclic peptides is important to evaluate possible conformations prior to synthesis. Because of the conformational constraints imposed by cyclization low energy conformations of cyclic peptides can be separated by large energy barriers. In order to improve the conformational search properties of molecular dynamics (MD) simulations a potential scaling method has been designed. The approach consists of several consecutive MD simulations with a specific lowering of dihedral energy barriers and reduced nonbonded interactions between atoms separated by three atoms followed by gradually scaling the potential until the original barriers are reached. Application to four cyclic penta- and hexa-peptide test cases and a protein loop of known structure indicates that the potential scaling method is more efficient and faster in locating low energy conformations than standard MD simulations. Combined with a generalized Born implicit solvation model the low energy cyclic peptide conformations and the loop structure are in good agreement with experiment. Applications in the presence of explicit water molecules during the simulations showed also improved convergence to structures close to experiment compared with regular MD.     

Characterization of the conformational behavior of peptide Contryphan Vn: a theoretical study

In this work we report the study of a peptide, the Contryphan Vn produced by Conus ventricosus, a vermivorous cone snail living in the temperate Mediterranean sea. This cyclic peptide of nine residues is a ring closed by a Cys-Cys (Cys: cysteine) disulfide bond containing two proline (Pro) residues and two tryptophans (Trp), one of them being a D-Trp. We present a statistical mechanical characterization of the peptide, simulated in water for about 200 ns with classical molecular dynamics (MD). In recent years there has been a growing interest in the study of the mechanics and dynamics of biological molecules, and in particular for proteins and peptides, about the relationship between collective motions and the active conformations which exert the biological function. To this aim we used the essential dynamics analysis on the MD trajectory and extracted, from the total fluctuations of the molecule, the dominant dynamical modes responsible of the principal conformational transitions. The Contryphan Vn small size allowed us to investigate in details the all-atoms dynamics and the corresponding thermodynamics in conformational space defined by the most significant intramolecular motions.     

Fluorescence study of domain structure and lipid interaction of human apolipoproteins E3 and E4

Human apolipoprotein E (apoE) isoforms exhibit different conformational stabilities and lipid-binding properties that give rise to altered cholesterol metabolism among the isoforms. Using Trp-substituted mutations and site-directed fluorescence labeling, we made a comprehensive comparison of the conformational organization of the N- and C-terminal domains and lipid interactions between the apoE3 and apoE4 isoforms. Trp fluorescence measurements for selectively Trp-substituted variants of apoE isoforms demonstrated that apoE4 adopts less stable conformations in both the N- and C-terminal domains compared to apoE3. Consistent with this, the conformational reorganization of the N-terminal helix bundle occurs at lower guanidine hydrochloride concentration in apoE4 than in apoE3 as monitored by fluorescence resonance energy transfer (FRET) from Trp residues to acrylodan attached at the N-terminal helix. Upon binding of apoE3 and apoE4 variants to egg phosphatidylcholine small unilamellar vesicles, similar changes in Trp fluorescence or FRET efficiency were observed for the isoforms, indicating that the opening of the N-terminal helix bundle occurs similarly in apoE3 and apoE4. Introduction of mutations into the C-terminal domain of the apoE isoforms to prevent self-association and maintain the monomeric state resulted in great increase in the rate of binding of the C-terminal helices to a lipid surface. Overall, our results demonstrate that the different conformational organizations of the N- and C-terminal domains have a minor effect on the steady-state lipid-binding behavior of apoE3 and apoE4: rather, self-association property is a critical determinant in the kinetics of lipid binding through the C-terminal helices of apoE isoforms.     

Ca2+- and S1-induced conformational changes of reconstituted skeletal muscle thin filaments observed by fluorescence energy transfer spectroscopy: structural evidence for three States of thin filament

Rabbit skeletal muscle alpha-tropomyosin (Tm) and the deletion mutant (D234Tm) in which internal actin-binding pseudo-repeats 2, 3, and 4 are missing [Landis et al. (1997) J. Biol. Chem. 272, 14051-14056] were used to investigate the interaction between actin and tropomyosin or actin and troponin (Tn) by means of fluorescence resonance energy transfer (FRET). FRET between Cys-190 of D234Tm and Gln-41 or Cys-374 of actin did not cause any significant Ca2+-induced movement of D234Tm, as reported previously for native Tm [Miki et al. (1998) J. Biochem. 123, 1104-1111]. FRET did not show any significant S1-induced movement of Tm and D234Tm on thin filaments either. The distances between Cys-133 of TnI, and Gln-41 and Cys-374 of actin on thin filaments reconstituted with D234Tm (mutant thin filaments) were almost the same as those on thin filaments with native Tm (wild-type thin filaments) in the absence of Ca2+. Upon binding of Ca2+ to TnC, these distances on mutant thin filaments increased by approximately 10 A in the same way as on wild-type thin filaments, which corresponds to a Ca2+-induced conformational change of thin filaments [Miki et al. (1998) J. Biochem. 123, 324-331]. The rigor binding of myosin subfragment 1 (S1) further increased these distances by approximately 7 A on both wild-type and mutant thin filaments when the thin filaments were fully decorated with S1. This indicates that a further conformational change on thin filaments was induced by S1 rigor-binding (S1-induced or open state). Plots of the extent of S1-induced conformational change vs. molar ratio of S1 to actin showed that the curve for wild-type thin filaments is hyperbolic, whereas that for mutant thin filaments is sigmoidal. This suggests that the transition to the S1-induced state on mutant thin filaments is depressed with a low population of rigor S1. In the absence of Ca2+, the distance also increased on both wild-type and mutant thin filaments close to the level in the presence of Ca2+ as the molar ratio of S1 to actin increased up to 1. The curves are sigmoidal for both wild-type and mutant thin filaments. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding. For mutant thin filaments, the transition from the closed state to the open state in the presence of ATP is strongly depressed, which results in the inhibition of acto-myosin ATPase even in the presence of Ca2+. The present FRET measurements provide structural evidence for three states of thin filaments (relaxed, Ca2+-induced or closed, and S1-induced or open states) for the regulation mechanism of skeletal muscle contraction.     

Synthetic and conformational studies on dehydroalanine-containing model peptides

Three model dipeptides containing a dehydroalanine residue (delta Ala) at the C-terminal, Boc-X-delta Ala-NHCH3 [X = Ala, Val, and Phe,] have been synthesized and their solution conformations investigated by 1H-NMR, IR, and CD spectroscopy. NMR studies on these peptides in CDCl3 clearly indicate that the NH group of dehydroalanine is involved in an intramolecular hydrogen bond. This conclusion is supported by IR studies also. Nuclear Overhauser effect (NOE) studies are also accommodative of an inverse gamma-turn-type of conformation that is characterized by conformational angles of phi approximately -70 degrees and psi approximately +70 degrees around the X residue, and a C alpha i + 1 H-Ni + 2H interproton distance of 2.5 A. It appears that unlike dehydrophenylalanine or dehydroleucine, which tend to stabilize beta-turn type of structures occupying the i + 2 position of the turn, dehydroalanine favors the formation of an inverse gamma-turn, centered at the preceding L-residue in such solvents as CDCl3 and (CD3)2SO. A comparison of solution conformation of Boc Val-delta Ala-NHCH3 with the corresponding saturated analogue, Boc-Val-Ala-NHCH3, is also presented and shows that dehydroalanine is responsible for inducing the turn structure. It may be possible to design peptides with different preferred conformations using the suitable dehydroamino acid.     

Direct sugar binding to LacY measured by resonance energy transfer

Trp151 in the lactose permease of Escherichia coli (LacY) is an important component of the sugar-binding site and the only Trp residue out of six that is in close proximity to the galactopyranoside in the structure (1PV7). The short distance between Trp151 and the sugar is favorable for F?rster resonance energy transfer (FRET) to nitrophenyl or dansyl derivatives with the fluorophore at the anomeric position of galactose. Modeling of 4-nitrophenyl-alpha-d-galactopyranoside (alpha-NPG) in the binding-site of LacY places the nitrophenyl moiety about 12 A away from Trp151, a distance commensurate with the F?rster distance for a Trp-nitrobenzoyl pair. We demonstrate here that alpha-NPG binding to LacY containing all six native Trp residues causes galactopyranoside-specific FRET from Trp151. Moreover, binding of alpha-NPG is sufficiently slow to resolve time-dependent fluorescence changes by stopped-flow. The rate of change in Trp --> alpha-NPG FRET is linearly dependent upon sugar concentration, which allows estimation of kinetic parameters for binding. Furthermore, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) covalently attached to the cytoplasmic end of helix X is sensitive to sugar binding, reflecting a ligand-induced conformational change. Stopped-flow kinetics of Trp --> alpha-NPG FRET and sugar-induced changes in MIANS fluorescence in the same protein reveal a two-step process: a relatively rapid binding step detected by Trp151 --> alpha-NPG FRET followed by a slower conformational change detected by a change in MIANS fluorescence.     

Sustained activation of AMP-activated protein kinase induces c-Jun N-terminal kinase activation and apoptosis in liver cells

We report the first Fourier transform infrared analysis of prion protein (PrP) repeats and the first study of PrP repeats of marsupial origin. Large changes in the secondary structure and an increase in hydrogen bonding within the peptide groups were evident from a red shift of the amide I band by >7 cm(-1) and an approximately five-fold reduction in amide hydrogen-deuterium exchange for peptide interacting with Cu(2+) ions. Changes in the tertiary structure upon copper binding were also evident from the appearance of a new band at 1564 cm(-1), which arises from the ring vibration of histidine. The copper-induced conformational change is pH dependent, and occurs at pH >7.     

Intra- and interspecies interactions between prion proteins and effects of mutations and polymorphisms

Recently, crystallization of the prion protein in a dimeric form was reported. Here we show that native soluble homogeneous FLAG-tagged prion proteins from hamster, man and cattle expressed in the baculovirus system are predominantly dimeric. The PrP/PrP interaction was confirmed in Semliki Forest virus-RNA transfected BHK cells co-expressing FLAG- and oligohistidine-tagged human PrP. The yeast two-hybrid system identified the octarepeat region and the C-terminal structured domain (aa90-aa230) of PrP as PrP/PrP interaction domains. Additional octarepeats identified in patients suffering from fCJD reduced (wtPrP versus PrP + 9OR) and completely abolished (PrP + 9OR versus PrP + 9OR) the PrP/PrP interaction in the yeast two-hybrid system. In contrast, the Met/Val polymorphism (aa129), the GSS mutation Pro102Leu and the FFI mutation Asp178Asn did not affect PrP/PrP interactions. Proof of interactions between human or sheep and bovine PrP, and sheep and human PrP, as well as lack of interactions between human or bovine PrP and hamster PrP suggest that interspecies PrP interaction studies in the yeast two-hybrid system may serve as a rapid pre-assay to investigate species barriers in prion diseases.     

Conformational study on glycosylated asparagine-oligopeptides by NMR spectroscopy and molecular dynamics calculations.

The conformational properties of the homo oligomers of increasing chain length Boc-(Asn)(n)-NHMe (n = 2, 4, 5), (GlcNAc-beta-Asn)(n)-NHMe (n = 2, 4, 5, 8) and Boc-[GlcNAc(Ac)(3)-beta-Asn](n)-NHMe (n = 2, 4, 5) were studied by using NOE experiments and molecular dynamic calculations (MD). Sequential NOEs and medium range NOEs, including (i,i+2) interactions, were detected by ROESY experiments and quantified. The calculated inter-proton distances are longer than those characteristic of beta-turn secondary structures. Owing to the large conformational motions expected for linear peptides, MD simulations were performed without NMR constraints, with explicit water and by applying different treatments of the electrostatic interactions. In agreement with the NOE results, the simulations showed, for all peptides, the presence of both folded and unfolded structures. The existence of significant populations of beta-turn structures can be excluded for all the examined compounds, but two families of structures were more often recognized. The first one with sinusoidal or S-shaped forms, and another family of large turns together with some more extended conformations. Only the glycosylated pentapeptide shows in vacuo a large amount of structures with helical shaped form. The results achieved in water and in DMSO are compared and discussed, together with the effect of the glycosylation.     

Ablation of the metal ion-induced endocytosis of the prion protein by disease-associated mutation of the octarepeat region.

The neurodegenerative spongiform encephalopathies, or prion diseases, are characterized by the conversion of the normal cellular form of the prion protein PrP(C) to a pathogenic form, PrP(Sc) [1]. There are four copies of an octarepeat PHGG(G/S)WGQ that specifically bind Cu(2+) ions within the N-terminal half of PrP(C) [2--4]. This has led to proposals that prion diseases may, in part, be due to abrogation of the normal cellular role of PrP(C) in copper homeostasis [5]. Here, we show that murine PrP(C) is rapidly endocytosed upon exposure of neuronal cells to physiologically relevant concentrations of Cu(2+) or Zn(2+), but not Mn(2+). Deletion of the four octarepeats or mutation of the histidine residues (H68/76 dyad) in the central two repeats abolished endocytosis, indicating that the internalization of PrP(C) is governed by metal binding to the octarepeats. Furthermore, a mutant form of PrP that contains nine additional octarepeats and is associated with familial prion disease [6] failed to undergo Cu(2+)-mediated endocytosis. For the first time, these results provide evidence that metal ions can promote the endocytosis of a mammalian prion protein in neuronal cells and that neurodegeneration associated with some prion diseases may arise from the ablation of this function due to mutation of the octarepeat region.