Two-site binding of beta-cyclodextrin to the Alzheimer Abeta(1-40) peptide measured with combined PFG-NMR diffusion and induced chemical shifts

The interactions of Alzheimer's amyloid beta-peptide with cyclodextrins were studied by (1)H NMR: the translational diffusion coefficient of the peptide and chemical shift changes were studied by the presence of variable concentrations of cyclodextrins. For the full-length peptide, Abeta(1-40), the combined results of translational diffusion and chemical shift changes are consistent with a model where aromatic side chains interact with beta-cyclodextrin with dissociation constants in the millimolar range. The diffusion data were consistent with two beta-cyclodextrin molecules bound per peptide. The binding occurs at two sites, at F(19) and/or F(20) and at Y(10), with dissociation constants K(d)(F) = 4.7 mM and K(d)(Y) = 6.6 mM, respectively, in 10 mM sodium phosphate, pH 7.4 and 298 K. Shorter Alzheimer peptide fragments were studied to measure specific affinities for different binding sites. The N-terminal fragment Abeta(1-9) with a putative binding site at F(4) does not show measurable affinity for beta-cyclodextrin. The fragment Abeta(12-28) has similar apparent affinity (K(d) = 3.8 mM) to beta-cyclodextrin as the full-length peptide Abeta(1-40). Here, the diffusion data suggests a one-to-one stoichiometry, and the binding site is F(19) and/or F(20). Both diffusion results and chemical shift changes give the same affinity. A variant Abeta(12-28)G(19)G(20) without phenylalanines does not bind to beta-cyclodextrin. Other potential ligands, alpha-cyclodextrin, gamma-cyclodextrin, nicotine, and nornicotine do not bind to the Abeta(12-28) fragment. This study shows that combined (1)H NMR diffusion and chemical shift changes may be used to quantitatively determine affinities and stoichiometries of weak interactions, using unlabeled ligands and hosts of comparable sizes.     

Structure of the 21-30 fragment of amyloid beta-protein

Folding and self-assembly of the 42-residue amyloid beta-protein (Abeta) are linked to Alzheimer's disease (AD). The 21-30 region of Abeta, Abeta(21-30), is resistant to proteolysis and is believed to nucleate the folding of full-length Abeta. The conformational space accessible to the Abeta(21-30) peptide is investigated by using replica exchange molecular dynamics simulations in explicit solvent. Conformations belonging to the global free energy minimum (the "native" state) from simulation are in good agreement with reported NMR structures. These conformations possess a bend motif spanning the central residues V24-K28. This bend is stabilized by a network of hydrogen bonds involving the side chain of residue D23 and the amide hydrogens of adjacent residues G25, S26, N27, and K28, as well as by a salt bridge formed between side chains of K28 and E22. The non-native states of this peptide are compact and retain a native-like bend topology. The persistence of structure in the denatured state may account for the resistance of this peptide to protease degradation and aggregation, even at elevated temperatures.     

On the nucleation of amyloid beta-protein monomer folding

Neurotoxic assemblies of the amyloid beta-protein (Abeta) have been linked strongly to the pathogenesis of Alzheimer's disease (AD). Here, we sought to monitor the earliest step in Abeta assembly, the creation of a folding nucleus, from which oligomeric and fibrillar assemblies emanate. To do so, limited proteolysis/mass spectrometry was used to identify protease-resistant segments within monomeric Abeta(1-40) and Abeta(1-42). The results revealed a 10-residue, protease-resistant segment, Ala21-Ala30, in both peptides. Remarkably, the homologous decapeptide, Abeta(21-30), displayed identical protease resistance, making it amenable to detailed structural study using solution-state NMR. Structure calculations revealed a turn formed by residues Val24-Lys28. Three factors contribute to the stability of the turn, the intrinsic propensities of the Val-Gly-Ser-Asn and Gly-Ser-Asn-Lys sequences to form a beta-turn, long-range Coulombic interactions between Lys28 and either Glu22 or Asp23, and hydrophobic interaction between the isopropyl and butyl side chains of Val24 and Lys28, respectively. We postulate that turn formation within the Val24-Lys28 region of Abeta nucleates the intramolecular folding of Abeta monomer, and from this step, subsequent assembly proceeds. This model provides a mechanistic basis for the pathologic effects of amino acid substitutions at Glu22 and Asp23 that are linked to familial forms of AD or cerebral amyloid angiopathy. Our studies also revealed that common C-terminal peptide segments within Abeta(1-40) and Abeta(1-42) have distinct structures, an observation of relevance for understanding the strong disease association of increased Abeta(1-42) production. Our results suggest that therapeutic approaches targeting the Val24-Lys28 turn or the Abeta(1-42)-specific C-terminal fold may hold promise.     

Attenuated cytotoxicity but enhanced betafibril of a mutant amyloid beta-peptide with a methionine to cysteine substitution

Amyloid-beta peptide (Abeta), the major constituent of senile plaques in the Alzheimer's disease (AD) brain, is the main source of oxidative stress leading to neurodegeneration. The methionine residue in this peptide is reported to be responsible for neurotoxicity. Structurally similar substitution with methionine 35 replaced by cysteine in Abeta(40) was synthesized, and this result in enhanced beta-sheet structures according to both circular dichroism (CD) spectra and beta-fibril specific fluorescence assay but attenuated cytotoxicity whether in the presence of copper or not. These findings may provide further evidence on disclosing the connection between amyloid beta-aggregation and Abeta-induced neurotoxicity.     

Interaction of angiotensin II with the C-terminal 300-320 fragment of the rat angiotensin II receptor AT1a monitored by NMR

Interaction between angiotensin II (Ang II) and the fragment peptide 300-320 (fCT300-320) of the rat angiotensin II receptor AT1a was demonstrated by relaxation measurements, NOE effects, chemical shift variations, and CD measurements. The correlation times modulating dipolar interactions for the bound and free forms of Ang II were estimated by the ratio of the nonselective and single-selective longitudinal relaxation rates. The intermolecular NOEs observed in NOESY spectra between HN protons of 9Lys(fCT) and 6His(ang), 10Phe(fCT) and 8Phe(ang), HN proton of 3Tyr(fCT) and Halpha of 4Tyr(ang), 5Phe(fCT)Hdelta and Halpha of 4Tyr(ang) indicated that Ang II aromatic residues are directly involved in the interaction, as also verified by relaxation data. Some fCT300-320 backbone features were inferred by the CSI method and CD experiments revealing that the presence of Ang II enhances the existential probability of helical conformations in the fCT fragment. Restrained molecular dynamics using the simulated annealing protocol was performed with intermolecular NOEs as constraints, imposing an alpha-helix backbone structure to fCT300-320 fragment. In the built model, one strongly preferred interaction was found that allows intermolecular stacking between aromatic rings and forces the peptide to wrap around the 6Leu side chain of the receptor fragment.     

Secondary structure conversions of Alzheimer's Abeta(1-40) peptide induced by membrane-mimicking detergents

The amyloid beta peptide (Abeta) with 39-42 residues is the major component of amyloid plaques found in brains of Alzheimer's disease patients, and soluble oligomeric peptide aggregates mediate toxic effects on neurons. The Abeta aggregation involves a conformational change of the peptide structure to beta-sheet. In the present study, we report on the effect of detergents on the structure transitions of Abeta, to mimic the effects that biomembranes may have. In vitro, monomeric Abeta(1-40) in a dilute aqueous solution is weakly structured. By gradually adding small amounts of sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate to a dilute aqueous solution, Abeta(1-40) is converted to beta-sheet, as observed by CD at 3 degrees C and 20 degrees C. The transition is mainly a two-state process, as revealed by approximately isodichroic points in the titrations. Abeta(1-40) loses almost all NMR signals at dodecyl sulfate concentrations giving rise to the optimal beta-sheet content (approximate detergent/peptide ratio = 20). Under these conditions, thioflavin T fluorescence measurements indicate a maximum of aggregated amyloid-like structures. The loss of NMR signals suggests that these are also involved in intermediate chemical exchange. Transverse relaxation optimized spectroscopy NMR spectra indicate that the C-terminal residues are more dynamic than the others. By further addition of SDS or lithium dodecyl sulfate reaching concentrations close to the critical micellar concentration, CD, NMR and FTIR spectra show that the peptide rearranges to form a micelle-bound structure with alpha-helical segments, similar to the secondary structures formed when a high concentration of detergent is added directly to the peptide solution.     

Oligopeptide-mediated acceleration of amyloid fibril formation of amyloid beta(Abeta) and alpha-synuclein fragment peptide (NAC).

The effects of oligopeptides on the secondary structures of Abeta and NAC, a fragment of alpha-synuclein protein, were studied by circular dichroism (CD) spectra. The effects of oligopeptides on the amyloid fibril formation were also studied by fluorescence spectra due to thioflavine-T. The oligopeptides were composed of a fragment of Abeta or NAC and were interposed by acidic or basic amino acid residues. The peptide, Ac-ELVFFAKK-NH2, which involved a fragment Leu-Val-Phe-Phe-Ala at Abeta(17-21), had no effect on the secondary structures of Abeta(1-28) in 60% or 90% trifluoroethanol (TFE) solutions at both pH 3.2 and pH 7.2. However, it showed pronounced effects on the secondary structure of Abeta(1-28) at pH 5.4. The Ac-ELVFFAKK-NH2 reduced the alpha-helical content, while it increased the beta-sheet content of Abeta(1-28). In phosphate buffer solutions at pH 7.0, Ac-ELVFFAKK-NH2 had little effect on the secondary structures of Abeta(1-28). However, it accelerated amyloid fibril formation when monitored by fluorescence spectra due to thioflavine-T. On the other hand, LPFFD, a peptide known as a beta-sheet breaker, caused neither an appreciable extent of change in the secondary structure nor amyloid fibril formation in the same buffer solution. The peptide, Ac-ETVK-NH2, which involved a fragment Thr-Val at NAC(21-22), had no effect on the secondary structure of NAC in 90% TFE and in isotonic phosphate buffer. However, Ac-ETVK-NH2 in water with small amounts of NaN3 and hexafluoroisopropanol greatly increased the beta-sheet content of NAC after standing the solution for more than 1 week. Interestingly, in this solution. Ac-ETVK-NH2, accelerated the fibril formation of NAC. It was concluded that an oligopeptide that involves a fragment of amyloidogenic proteins could be a trigger for the formation of amyloid plaques of the proteins even when it had little effect on the secondary structures of the proteins as monitored by CD spectra for a short incubation time.     

Conformational solution studies of the SDS micelle-bound 11-28 fragment of two Alzheimer's beta-amyloid variants (E22K and A21G) using CD, NMR, and MD techniques

The beta-amyloid (Abeta) is the major peptide constituent of neuritic plaques in Alzheimer's disease (AD) and its aggregation is believed to play a central role in the pathogenesis of the disease. Naturally occurring mutations resulting in changes in the Abeta sequence (pos. 21-23) are associated with familial AD-like diseases with extensive cerebrovascular pathology. It was proved that the mutations alter the aggregation ability of Abeta and its neurotoxicity. Among five mutations at positions 21-23 there are two mutations with distinct clinical characteristics and potentially distinct pathogenic mechanism-the Italian (E22K) and the Flemish (A21G) mutations. In our studies we have examined the structures of the 11-28 fragment of the Italian and Flemish Abeta variants. The fragment was chosen because it has been shown to be the most important for amyloid fibril formation. The detailed structure of both variants Abeta(11-28) was determined using CD, 2D NMR, and molecular dynamics techniques under water-SDS micelle conditions. The NMR analysis revealed two distinct sets of proton resonances for the peptides. The studies of both peptides pointed out the existence of well-defined alpha-helical conformation in the Italian mutant, whereas the Flemish was found to be unstructured with the possibility of a bent structure in the central part of the peptide.     

Stabilization of discordant helices in amyloid fibril-forming proteins

Several proteins and peptides that can convert from alpha-helical to beta-sheet conformation and form amyloid fibrils, including the amyloid beta-peptide (Abeta) and the prion protein, contain a discordant alpha-helix that is composed of residues that strongly favor beta-strand formation. In their native states, 37 of 38 discordant helices are now found to interact with other protein segments or with lipid membranes, but Abeta apparently lacks such interactions. The helical propensity of the Abeta discordant region (K16LVFFAED23) is increased by introducing V18A/F19A/F20A replacements, and this is associated with reduced fibril formation. Addition of the tripeptide KAD or phospho-L-serine likewise increases the alpha-helical content of Abeta(12-28) and reduces aggregation and fibril formation of Abeta(1-40), Abeta(12-28), Abeta(12-24), and Abeta(14-23). In contrast, tripeptides with all-neutral, all-acidic or all-basic side chains, as well as phosphoethanolamine, phosphocholine, and phosphoglycerol have no significant effects on Abeta secondary structure or fibril formation. These data suggest that in free Abeta, the discordant alpha-helix lacks stabilizing interactions (likely as a consequence of proteolytic removal from a membrane-associated precursor protein) and that stabilization of this helix can reduce fibril formation.     

Folding landscapes of the Alzheimer amyloid-beta(12-28) peptide

The energy landscape for folding of the 12-28 fragment of the Alzheimer amyloid beta (Abeta) peptide is characterized using replica-exchange molecular dynamics simulations with an all-atom peptide model and explicit solvent. At physiological temperatures, the peptide exists mostly as a collapsed random coil, populating a small fraction (less than 10%) of hairpins with a beta-turn at position V18F19, with another 10% of hairpin-like conformations possessing a bend rather than a turn in the central VFFA positions. A small fraction of the populated states, approximately 14%, adopt polyproline II (PPII) conformations. Folding of the structured hairpin states proceeds through the assembly of two locally stable segments, VFFAE and EDVGS. The interactions stabilizing these locally folded structural motifs are in conflict with those stabilizing the global fold of A12-28, a signature of underlying residual frustration in this peptide. At increased temperature, the population of both beta-strand and PPII conformations diminishes in favor of beta-turn and random-coil states. On the basis of the conformational preferences of Abeta 12-28 monomers, two models for the molecular structure of amyloid fibrils formed by this peptide are proposed.     

Comparison of the structures of beta amyloid peptide (25-35) and substance P in trifluoroethanol/water solution

The three-dimensional structure of beta amyloid peptide (25-35) in aqueous solution with 50% (vol/vol) 2,2,2-trifluoroethanol was determined by NMR spectroscopy. Beta amyloid peptide(Abeta) is the major component of senile plaques found in the brain of patient of Alzheimer's disease. Abeta25-35 is biologically active fragment of Abeta and exhibits some sequence homology with the tachykinin family. In this study, we present the structural similarity between Abeta25-35 and substance P which is a member of tachykinin family in order to examine the possibility of sharing pathways mediated by tachykinin receptors. Both peptides have alpha-helical structures in their C-terminal regions and aromatic rings or hydrophobic side chains in the center of the helix protrude outside. These conformational features are expected to be the key for the interaction with the receptors.     

A left-handed 3(1) helical conformation in the Alzheimer Abeta(12-28) peptide

We show for the first time that the secondary structure of the Alzheimer beta-peptide is in a temperature-dependent equilibrium between an extended left-handed 3(1) helix and a flexible random coil conformation. Circular dichroism spectra, recorded at 0.03 mM peptide concentration, show that the equilibrium is shifted towards increasing left-handed 3(1) helix structure towards lower temperatures. High resolution nuclear magnetic resonance (NMR) spectroscopy has been used to study the Alzheimer peptide fragment Abeta(12-28) in aqueous solution at 0 degrees C and higher temperatures. NMR translation diffusion measurements show that the observed peptide is in monomeric form. The chemical shift dispersion of the amide protons increases towards lower temperatures, in agreement with the increased population of a well-ordered secondary structure. The solvent exchange rates of the amide protons at 0 degrees C and pH 4.5 vary within at least two orders of magnitude. The lowest exchange rates (0.03-0.04 min(-1)) imply that the corresponding amide protons may be involved in hydrogen bonding with neighboring side chains.     

Deposition of monomeric, not oligomeric, Abeta mediates growth of Alzheimer's disease amyloid plaques in human brain preparations.

Senile plaques composed of the peptide Abeta contribute to the pathogenesis of Alzheimer's disease (AD), and mechanisms underlying their formation and growth may be exploitable as therapeutic targets. To examine the process of amyloid plaque growth in human brain, we have utilized size exclusion chromatography (SEC), translational diffusion measured by NMR, and in vitro models of Abeta amyloid growth to identify the oligomerization state of Abeta that is competent to add onto an existing amyloid deposit. SEC of radiolabeled and unlabeled Abeta over a concentration range of 10(-)(10)-10(-)(4) M demonstrated that the freshly dissolved peptide eluted as a single low molecular weight species, consistent with monomer or dimer. This low molecular weight Abeta species isolated by SEC was competent to deposit onto preexisting amyloid in preparations of AD cortex, with first-order kinetic dependence on soluble Abeta concentration, establishing that solution-phase oligomerization is not rate limiting. Translational diffusion measurements of the low molecular weight Abeta fraction demonstrate that the form of the peptide active in plaque deposition is a monomer. In deliberately aged (>6 weeks) Abeta solutions, a high molecular weight (>100 000 M(r)) species was detectable in the SEC column void. In contrast to the active monomer, assembled Abeta isolated from the column showed little or no focal association with AD tissue. These studies establish that, at least in vitro, Abeta exists as a monomer at physiological concentrations and that deposition of monomers, rather than of oligomeric Abeta assemblies, mediates the growth of existing amyloid in human brain preparations.     

Aromatic interactions in model peptide β-hairpins: ring current effects on proton chemical shifts

Crystal structures of eight peptide β-hairpins in the sequence Boc-Leu-Phe-Val-Xxx-Yyy-Leu-Phe-Val-OMe revealed that the Phe(2) and Phe(7) aromatic rings are in close spacial proximity, with the centroid-centroid distance (R(cen)) of 4.4-5.4 ? between the two phenyl rings. Proton NMR spectra in chloroform and methanol solution reveal a significant upfield shift of the Phe(7) C(δ,δ') H(2) protons (6.65-7.04 ppm). Specific assignments of the aromatic protons have been carried out in the peptide Boc-Leu-Phe-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe (6). The anticipated ring current shifts have been estimated from the aromatic ring geometrics observed in crystals for all eight peptides. Only one of the C(δ,δ') H proton lies in the shielding zone with rapid ring flipping, resulting in averaging between the two extreme chemical shifts. An approximate estimate of the population of conformations, which resemble crystal state orientation, may be obtained. Key nuclear Overhauser effects (NOEs) between facing Phe side chains provide support for close similarity between the solid state and solution conformation. Temperature dependence of aromatic ring proton chemical shift and line widths for peptide 6 (Boc-Leu-Phe-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe) and the control peptide Boc-Leu-Val-Val-(D)Pro-Gly-Leu-Phe-Val-OMe establish an enhanced barrier to ring flipping when the two Phe rings are in proximity. Modeling studies suggest that small, conformational adjustment about C(α)-C(β) (χ(1) ) and C(β)-C(γ) (χ(2) ) bonds of both the Phe residues may be required in order to permit unhindered, uncorrelated flipping of both the Phe rings. The maintenance of the specific aromatic ring orientation in organic solvents provides evidence for significant stabilizing interaction.     

Characterizations of distinct amyloidogenic conformations of the Abeta (1-40) and (1-42) peptides

Major constituents of the amyloid plaques found in the brain of Alzheimer's patients are the 39-43 residue beta-amyloid (Abeta) peptides. Extensive in vitro as well as in vivo biochemical studies have shown that the 40- and 42-residue Abeta peptides play major roles in the neurodegenerative pathology of Alzheimer's disease. Although the two Abeta peptides share common aggregation properties, the 42-residue peptide is more amyloidogenic and more strongly associated with amyloid pathology. Thus, characterizations of the two Abeta peptides are of critical importance in understanding the molecular mechanism of Abeta amyloid formation. In this report, we present combined CD and NMR studies of the monomeric states of the two peptides under both non-amyloidogenic (<5 degrees C) and amyloid-forming conditions (>5 degrees C) at physiological pH. Our CD studies of the Abeta peptides showed that initially unfolded Abeta peptides at low temperature (<5 degrees C) gradually underwent conformational changes to more beta-sheet-like monomeric intermediate states at stronger amyloidogenic conditions (higher temperatures). Detailed residue-specific information on the structural transition was obtained by using NMR spectroscopy. Residues in the N-terminal (3-12) and 20-22 regions underwent conformational changes to more extended structures at the stronger amyloidogenic conditions. Almost identical structural transitions of those residues were observed in the two Abeta peptides, suggesting a similar amyloidogenic intermediate for the two peptides. The 42-residue Abeta (1-42) peptide was, however, more significantly structured at the C-terminal region (39-42), which may lead to the different aggregation propensity of the two peptides.     

Controlling {beta}-amyloid oligomerization by the use of naphthalene sulfonates: trapping low molecular weight oligomeric species

Aggregation of proteins and peptides has been shown to be responsible for several diseases known as amyloidoses, which include Alzheimer disease (AD), prion diseases, among several others. AD is a neurodegenerative disorder caused primarily by the aggregation of beta-amyloid peptide (Abeta). Here we describe the stabilization of small oligomers of Abeta by the use of sulfonated hydrophobic molecules such as AMNS (1-amino-5-naphthalene sulfonate); 1,8-ANS (1-anilinonaphthalene-8-sulfonate) and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate). The experiments were performed with either Abeta-1-42 or with Abeta-13-23, a shorter version of Abeta that is still able to form amyloid fibrils in vitro and contains amino acid residues 16-20, previously shown to be essential to peptide-peptide interaction and fibril formation. All sulfonated molecules tested were able to prevent Abeta aggregation in a concentration dependent fashion in the following order of efficacy: 1,8-ANS < AMNS < bis-ANS. Size exclusion chromatography revealed that in the presence of bis-ANS, Abeta forms a heterogeneous population of low molecular weight species that proved to be toxic to cell cultures. Since the ANS compounds all have apolar rings and negative charges (sulfonate groups), both hydrophobic and electrostatic interactions may contribute to interpeptide contacts that lead to aggregation. We also performed NMR experiments to investigate the structure of Abeta-13-23 in SDS micelles and found features of an alpha-helix from Lys(16) to Phe(20). 1H TOCSY spectra of Abeta-13-23 in the presence of AMNS displayed a chemical-shift dispersion quite similar to that observed in SDS, which suggests that in the presence of AMNS this peptide might adopt a conformation similar to that reported in the presence of SDS. Taken together, our studies provide evidence for the crucial role of small oligomers and their stabilization by sulfonate hydrophobic compounds.     

Effects of grape seed-derived polyphenols on amyloid beta-protein self-assembly and cytotoxicity

Epidemiological evidence suggests that moderate consumption of red wine reduces the incidence of Alzheimer disease (AD). To study the protective effects of red wine, experiments recently were executed in the Tg2576 mouse model of AD. These studies showed that a commercially available grape seed polyphenolic extract, MegaNatural-AZ (MN), significantly attenuated AD-type cognitive deterioration and reduced cerebral amyloid deposition (Wang, J., Ho, L., Zhao, W., Ono, K., Rosensweig, C., Chen, L., Humala, N., Teplow, D. B., and Pasinetti, G. M. (2008) J. Neurosci. 28, 6388-6392). To elucidate the mechanistic bases for these observations, here we used CD spectroscopy, photo-induced cross-linking of unmodified proteins, thioflavin T fluorescence, size exclusion chromatography, and electron microscopy to examine the effects of MN on the assembly of the two predominant disease-related amyloid beta-protein alloforms, Abeta40 and Abeta42. We also examined the effects of MN on Abeta-induced cytotoxicity by assaying 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide metabolism and lactate dehydrogenase activity in Abeta-treated, differentiated pheochromocytoma (PC12) cells. Initial studies revealed that MN blocked Abeta fibril formation. Subsequent evaluation of the assembly stage specificity of the effect showed that MN was able to inhibit protofibril formation, pre-protofibrillar oligomerization, and initial coil --> alpha-helix/beta-sheet secondary structure transitions. Importantly, MN had protective effects in assays of cytotoxicity in which MN was mixed with Abeta prior to peptide assembly or following assembly and just prior to peptide addition to cells. These data suggest that MN is worthy of consideration as a therapeutic agent for AD.     

Experimental constraints on quaternary structure in Alzheimer's beta-amyloid fibrils

We describe solid-state nuclear magnetic resonance (NMR) measurements on fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease (Abeta(1-40)) that place constraints on the identity and symmetry of contacts between in-register, parallel beta-sheets in the fibrils. We refer to these contacts as internal and external quaternary contacts, depending on whether they are within a single molecular layer or between molecular layers. The data include (1) two-dimensional 13C-13C NMR spectra that indicate internal quaternary contacts between side chains of L17 and F19 and side chains of I32, L34, and V36, as well as external quaternary contacts between side chains of I31 and G37; (2) two-dimensional 15N-13C NMR spectra that indicate external quaternary contacts between the side chain of M35 and the peptide backbone at G33; (3) measurements of magnetic dipole-dipole couplings between the side chain carboxylate group of D23 and the side chain amine group of K28 that indicate salt bridge interactions. Isotopic dilution experiments allow us to make distinctions between intramolecular and intermolecular contacts. On the basis of these data and previously determined structural constraints from solid-state NMR and electron microscopy, we construct full molecular models using restrained molecular dynamics simulations and restrained energy minimization. These models apply to Abeta(1-40) fibrils grown with gentle agitation. We also present evidence for different internal quaternary contacts in Abeta(1-40) fibrils grown without agitation, which are morphologically distinct.     

Proton NMR studies of transforming and nontransforming H-ras p21 mutants

One- and two-dimensional nuclear magnetic resonance spectroscopy (1D and 2D NMR) and site-directed mutagenesis were used to study the influence of mutations on the conformation of the H-ras oncogene product p21. No severe structural differences between the different mutants, whether they were transforming or nontransforming, could be detected. Initially, selective incorporation of 3,5-deuterated tyrosyl residues into p21 and 2D NMR were used to identify the resonances representing the spin systems of the imidazole rings of the three histidyl residues in the protein, of six of the nine tyrosyl rings, and of four of the five phenylalanyl rings. The spin systems of the phenyl rings of Phe28, Phe78, and Phe82 could be assigned by using mutant proteins, since no severe structure-induced spectral changes in the aromatic part of the spectra of the mutant proteins were detected. Sequence-specific assignments of the histidine imidazole resonances could be obtained by comparison of the distance information obtained by nuclear Overhauser enhancement spectroscopy (NOESY) experiments with the crystal structure. The change in the chemical shift values of the Hl' proton and the alpha-phosphate of the bound GDP in the NMR spectra of the p21(F28L) mutant and the 28-fold increase in the GDP dissociation rate constants of this mutant suggest a strong interaction between Phe28 and the p21-bound nucleotide. In solution, the p21-bound GDP.Mg2+ has an anti conformation, and the phenyl ring of Phe28 is close to the ribose of the bound GDP.Mg2+.     

Probing aromatic, hydrophobic, and steric effects on the self-assembly of an amyloid-β fragment peptide

Aromatic amino acids have been shown to promote self-assembly of amyloid peptides, although the basis for this amyloid-inducing behavior is not understood. We adopted the amyloid-β 16-22 peptide (Aβ(16-22), Ac-KLVFFAE-NH(2)) as a model to study the role of aromatic amino acids in peptide self-assembly. Aβ(16-22) contains two consecutive Phe residues (19 and 20) in which Phe 19 side chains form interstrand contacts in fibrils while Phe 20 side chains interact with the side chain of Va l18. The kinetic and thermodynamic effect of varying the hydrophobicity and aromaticity at positions 19 and 20 by mutation with Ala, Tyr, cyclohexylalanine (Cha), and pentafluorophenylalanine (F(5)-Phe) (order of hydrophobicity is Ala < Tyr < Phe < F(5)-Phe < Cha) was characterized. Ala and Tyr position 19 variants failed to undergo fibril formation at the peptide concentrations studied, but Cha and F(5)-Phe variants self-assembled at dramatically enhanced rates relative to wild-type. Cha mutation was thermodynamically stabilizing at position 20 (ΔΔG = -0.2 kcal mol(-1) relative to wild-type) and destabilizing at position 19 (ΔΔG = +0.2 kcal mol(-1)). Conversely, F(5)-Phe mutations were strongly stabilizing at both positions (ΔΔG = -1.3 kcal mol(-1) at 19, ΔΔG = -0.9 kcal mol(-1) at 20). The double Cha and F(5)-Phe mutants showed that the thermodynamic effects were additive (ΔΔG = 0 kcal mol(-1) for Cha 19,20 and -2.1 kcal mol(-1) for F(5)-Phe 19,20). These results indicate that sequence hydrophobicity alone does not dictate amyloid potential, but that aromatic, hydrophobic, and steric considerations collectively influence fibril formation.