Sequence dependence of beta-hairpin structure: comparison of a salt bridge and an aromatic interaction

A comparison of the contributions and position dependence of cross-strand electrostatic and aromatic side-chain interactions to beta-sheet stability has been performed by using nuclear magnetic resonance in a well-folded beta-hairpin peptide of the general sequence XRTVXVdPGOXITQX. Phe-Phe and Glu-Lys pairs were varied at the internal and terminal non-hydrogen-bonded position, and the resulting stability was measured by the effects on alpha-hydrogen and aromatic hydrogen chemical shifts. It was determined that the introduction of a Phe-Phe pair resulted in a more folded peptide, regardless of position, and a more tightly folded core. Substitution of the Glu-Lys pair at the internal position results in a less folded peptide and increased fraying at the terminal residues. Upfield shifting of the aromatic hydrogens provided evidence for an edge-face aromatic interaction, regardless of position of the Phe-Phe pair. In peptides with two Phe-Phe pairs, substitution with Glu-Lys at either position resulted in a weakening of the aromatic interaction and a subsequent decrease in peptide stability. Thermal denaturation of the peptides containing Phe-Phe indicates that the aromatic interaction is enthalpically favored, whereas the folding of hairpins with cross-strand Glu-Lys pairs was less enthalpically favorable but entropically more favorable.     

A cross-strand Trp Trp pair stabilizes the hPin1 WW domain at the expense of function

Using the human Pin1 WW domain (hPin1 WW), we show that replacement of two nearest neighbor non-hydrogen-bonded residues on adjacent beta-strands with tryptophan (Trp) residues increases beta-sheet thermodynamic stability by 4.8 kJ mol(-1) at physiological temperature. One-dimensional NMR studies confirmed that introduction of the Trp-Trp pair does not globally perturb the structure of the triple-stranded beta-sheet, while circular dichroism studies suggest that the engineered cross-strand Trp-Trp pair adopts a side-chain conformation similar to that first reported for a designed "Trp-zipper" beta-hairpin peptide, wherein the indole side chains stack perpendicular to each other. Even though the mutated side chains in wild-type hPin1 WW are not conserved among WW domains and compose the beta-sheet surface opposite to that responsible for ligand binding, introduction of the cross-strand Trp-Trp pair effectively eliminates hPin1 WW function as assessed by the loss of binding affinity toward a natural peptide ligand. Maximizing both thermodynamic stability and the domain function of hPin1 WW by the above mentioned approach appears to be difficult, analogous to the situation with loop 1 optimization explored previously. That introduction of a non-hydrogen-bonded cross-strand Trp-Trp pair within the hPin1 WW domain eliminates function may provide a rationale for why this energetically favorable pairwise interaction has not yet been identified in WW domains or any other biologically evolved protein with known three-dimensional structure.     

Alzheimer's Disease: NMR Studies of Asialo (GM1) and Trisialo (GT1b) Ganglioside Interactions with Aβ(1-40) Peptide in a Membrane Mimic Environment

Amyloid peptide (Abeta) is the major protein constituent of neuritic plaques in Alzheimer's disease (AD). This peptide is an amphipathic molecule that perturbs membranes and binds to raft-like membranes composed of gangliosides. Ganglioside GM1 binds tightly with Abeta and it is speculated that GM1 inhibits Abeta from undergoing alpha-helix to beta-sheet conformational changes. Although the role of gangliosides in conformational changes of Abeta have been studied, the specific nature of these interactions have not been reported. In the present report multidimensional NMR studies of ganglioside-Abeta interactions were conducted in sodium dodecyl sulphate (SDS) micelles, a membrane-mimicking environment. These studies reveal that asialoGM1 binds specifically with Abeta in a manner which could prevent beta-sheet formation. but that ganglioside GT1b does not bind Abeta. Plausible pathways for the involvement of gangliosides in amyloidogenesis are discussed.     

Review: modulating factors in amyloid-beta fibril formation

Amyloid formation is a key pathological feature of Alzheimer's disease and is considered to be a major contributing factor to neurodegeneration and clinical dementia. Amyloid is found as both diffuse and senile plaques in the parenchyma of the brain and is composed primarily of the 40- to 42-residue amyloid-beta (Abeta) peptides. The characteristic amyloid fiber exhibits a high beta-sheet content and may be generated in vitro by the nucleation-dependent self-association of the Abeta peptide and an associated conformational transition from random to beta-conformation. Growth of the fibrils occurs by assembly of the Abeta seeds into intermediate protofibrils, which in turn self-associate to form mature fibers. This multistep process may be influenced at various stages by factors that either promote or inhibit Abeta fiber formation and aggregation. Identification of these factors and understanding the driving forces behind these interactions as well as the structural motifs necessary for these interactions will help to elucidate potential sites that may be targeted to prevent amyloid formation and its associated toxicity. This review will discuss some of the modulating factors that have been identified to date and their role in fibrillogenesis.     

The roles of turn formation and cross-strand interactions in fibrillization of peptides derived from the OspA single-layer beta-sheet

We previously demonstrated that a beta-hairpin peptide, termed BH(9-10), derived from a single-layer beta-sheet of Borrelia OspA protein, formed a native-like beta-turn in trifluoroethanol (TFE) solution, and it assembled into amyloid-like fibrils at higher TFE concentrations. This peptide is highly charged, and fibrillization of such a hydrophilic peptide is quite unusual. In this study, we designed a circularly permutated peptide of BH(9-10), termed BH(10-9). When folded into their respective beta-hairpin structures found in OspA, these peptides would have identical cross-strand interactions but different turns connecting the strands. NMR study revealed that BH(10-9) had little propensity to form a turn structure both in aqueous and TFE solutions. At higher TFE concentration, BH(10-9) precipitated with a concomitant alpha-to-beta conformational conversion, in a similar manner to the BH(9-10) fibrillization. However, the BH(10-9) precipitates were nonfibrillar aggregation. The precipitation kinetics of BH(10-9) was exponential, consistent with a first-order molecular assembly reaction, while the fibrillization of BH(9-10) showed sigmoidal kinetics, indicative of a two-step reaction consisting of nucleation and molecular assembly. The correlation between native-like turn formation and fibrillization of our peptide system strongly suggests that BH(9-10) adopts a native-like beta-hairpin conformation in the fibrils. Remarkably, seeding with the preformed BH(10-9) precipitates changed the two-step BH(9-10) fibrillization to a one-step molecular assembly reaction, and disrupted the BH(9-10) fibril structure, indicating interactions between the BH(10-9) aggregates and the BH(9-10) peptide. Our results suggest that, in these peptides, cross-strand interactions are the driving force for molecular assembly, and turn formation limits modes of peptide assembly.     

Probing the role of backbone hydrogen bonding in beta-amyloid fibrils with inhibitor peptides containing ester bonds at alternate positions

Protein-protein interactions are frequently mediated by stable, intermolecular beta-sheets. A number of cytokines and the HIV Protease, for example, dimerize through beta-sheet motifs. Evidence also suggests that the macromolecular assemblies of peptides and proteins in amyloid fibrils are stabilized by intermolecular beta-sheets. In this paper, we report that interfering with the backbone hydrogen bonding of an amyloidgenic peptide (Abeta16-20) by replacing amide bonds with ester bonds prevents the aggregation of the peptide. The ester bonds were incorporated in an alternating fashion so that the peptide presents two unique hydrogen bonding faces when arrayed in an extended, beta-strand conformation; one face of the peptide has normal hydrogen bonding capabilities, but the other face is missing amide protons and its ability to hydrogen bond is severely limited. Analytical ultracentrifugation experiments demonstrate that this ester peptide, Abeta16-20e, is predominantly monomeric under solution conditions, unlike the fibril-forming Abeta16-20 peptide. Abeta16-20e also inhibits the aggregation of the Abeta1-40 peptide and disassembles preformed Abeta1-40 fibrils. These results suggest that backbone hydrogen bonding is critical for the assembly of amyloid fibrils.     

Lipid membrane templates the ordering and induces the fibrillogenesis of Alzheimer's disease amyloid-beta peptide

The lipid membrane has been shown to mediate the fibrillogenesis and toxicity of Alzheimer's disease (AD) amyloid-beta (Abeta) peptide. Electrostatic interactions between Abeta40 and the phospholipid headgroup have been found to control the association and insertion of monomeric Abeta into lipid monolayers, where Abeta exhibited enhanced interactions with charged lipids compared with zwitterionic lipids. To elucidate the molecular-scale structural details of Abeta-membrane association, we have used complementary X-ray and neutron scattering techniques (grazing-incidence X-ray diffraction, X-ray reflectivity, and neutron reflectivity) in this study to investigate in situ the association of Abeta with lipid monolayers composed of either the anionic lipid 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), the zwitterionic lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or the cationic lipid 1,2-dipalmitoyl 3-trimethylammonium propane (DPTAP) at the air-buffer interface. We found that the anionic lipid DPPG uniquely induced crystalline ordering of Abeta at the membrane surface that closely mimicked the beta-sheet structure in fibrils, revealing an intriguing templated ordering effect of DPPG on Abeta. Furthermore, incubating Abeta with lipid vesicles containing the anionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) induced the formation of amyloid fibrils, confirming that the templated ordering of Abeta at the membrane surface seeded fibril formation. This study provides a detailed molecular-scale characterization of the early structural fluctuation and assembly events that may trigger the misfolding and aggregation of Abeta in vivo. Our results implicate that the adsorption of Abeta to anionic lipids, which could become exposed to the outer membrane leaflet by cell injury, may serve as an in vivo mechanism of templated-aggregation and drive the pathogenesis of AD.     

Insights into the determinants of beta-sheet stability: 1H and 13C NMR conformational investigation of three-stranded antiparallel beta-sheet-forming peptides.

In a previous study we designed a 20-residue peptide able to adopt a significant population of a three-stranded antiparallel beta-sheet in aqueous solution (de Alba et al. [1999]Protein Sci.8, 854-865). In order to better understand the factors contributing to beta-sheet folding and stability we designed and prepared nine variants of the parent peptide by substituting residues at selected positions in its strands. The ability of these peptides to form the target motif was assessed on the basis of NMR parameters, in particular NOE data and 13Calpha conformational shifts. The populations of the target beta-sheet motif were lower in the variants than in the parent peptide. Comparative analysis of the conformational behavior of the peptides showed that, as expected, strand residues with low intrinsic beta-sheet propensities greatly disfavor beta-sheet folding and that, as already found in other beta-sheet models, specific cross-strand side chain-side chain interactions contribute to beta-sheet stability. More interestingly, the performed analysis indicated that the destabilization effect of the unfavorable strand residues depends on their location at inner or edge strands, being larger at the latter. Moreover, in all the cases examined, favorable cross-strand side chain-side chain interactions were not strong enough to counterbalance the disfavoring effect of a poor beta-sheet-forming residue, such as Gly.     

Interactions of amyloid beta-peptide (1-40) with ganglioside-containing membranes

Interactions between amyloid beta-peptides (Abeta) and neuronal membranes have been postulated to play an important role in the neuropathology of Alzheimer's disease. To gain insight into the molecular details of this association, we investigated the interactions of Abeta (1-40) with ganglioside-containing membranes by circular dichroism (CD) and Fourier transform infrared-polarized attenuated total reflection (FTIR-PATR) spectroscopy. The CD study revealed that at physiological ionic strength Abeta (1-40) specifically binds to ganglioside-containing membranes inducing a two-state, unordered --> beta-sheet transition above a threshold intramembrane ganglioside concentration, which depends on the host lipid bilayers used. Furthermore, differences in the number and position of sialic acid residues of the carbohydrate backbone significantly affected the conformational transition of the peptide. FTIR-PATR spectroscopy experiments demonstrated that Abeta (1-40) forms an antiparallel beta-sheet, the plane of which lies parallel to the membrane surface, inducing dehydration of lipid interfacial groups and perturbation of acyl chain orientation. These results suggest that Abeta (1-40) imposes negative curvature strain on ganglioside-containing lipid bilayers, disturbing the structure and function of the membranes.     

Rapid assembly of amyloid-beta peptide at a liquid/liquid interface produces unstable beta-sheet fibers

Accumulation of aggregated amyloid-beta peptide (Abeta) in the brain is a pathological hallmark of Alzheimer's disease (AD). In vitro studies indicate that the 40- to 42-residue Abeta peptide in solution will undergo self-assembly leading to the transient appearance of soluble protofibrils and ultimately to insoluble fibrils. The Abeta peptide is amphiphilic and accumulates preferentially at a hydrophilic/hydrophobic interface. Solid surfaces and air-water interfaces have been shown previously to promote Abeta aggregation, but detailed characterization of these aggregates has not been presented. In this study Abeta(1-40) introduced to aqueous buffer in a two-phase system with chloroform aggregated 1-2 orders of magnitude more rapidly than Abeta in the buffer alone. The interface-induced aggregates were released into the aqueous phase and persisted for 24-72 h before settling as a visible precipitate at the interface. Thioflavin T fluorescence and circular dichroism analyses confirmed that the Abeta aggregates had a beta-sheet secondary structure. However, these aggregates were far less stable than Abeta(1-40) protofibrils prepared in buffer alone and disaggregated completely within 3 min on dilution. Atomic force microscopy revealed that the aggregates consisted of small globules 4-5 nm in height and long flexible fibers composed of these globules aligned roughly along a longitudinal axis, a morphology distinct from that of Abeta protofibrils prepared in buffer alone. The relative instability of the fibers was supported by fiber interruptions apparently introduced by brief washing of the AFM grids. To our knowledge, unstable aggregates of Abeta with beta-sheet structure and fibrous morphology have not been reported previously. Our results provide the clearest evidence yet that the intrinsic beta-sheet structure of an in vitro Abeta aggregate depends on the aggregation conditions and is reflected in the stability of the aggregate and the morphology observed by atomic force microscopy. Resolution of these structural differences at the molecular level may provide important clues to the further understanding of amyloid formation in vivo.     

Influence of hydrophobic Teflon particles on the structure of amyloid beta-peptide

The amyloid beta-protein (Abeta) constitutes the major peptide component of the amyloid plaque deposits of Alzheimer's disease in humans. The Abeta changes from a nonpathogenic to a pathogenic conformation resulting in self-aggregation and deposition of the peptide. It has been established that denaturing factors (such as the interaction with membranes) are involved in the structural transition. This work is aimed at determining the effect of hydrophobic Teflon on the conformation of the Abeta (1-40). Prior to adsorption, the secondary structure and self-aggregation state of the Abeta in solution were established as a function of pH. Three different species coexist: unordered monomers/dimers, small oligomers in mainly a regular beta-sheet structure, and bigger aggregates having a twisted beta-sheet conformation. Transferring the Abeta from the solution to the Teflon surface strongly promotes alpha-helix formation. Furthermore, increasing the degree of coverage of the Teflon by the Alphabeta protein leads to a conformational change toward a more enriched beta-sheet structure.     

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.     

Inhibitors of amyloid toxicity based on beta-sheet packing of Abeta40 and Abeta42

Amyloid fibrils associated with Alzheimer's disease and a wide range of other neurodegenerative diseases have a cross beta-sheet structure, where main chain hydrogen bonding occurs between beta-strands in the direction of the fibril axis. The surface of the beta-sheet has pronounced ridges and grooves when the individual beta-strands have a parallel orientation and the amino acids are in-register with one another. Here we show that in Abeta amyloid fibrils, Met35 packs against Gly33 in the C-terminus of Abeta40 and against Gly37 in the C-terminus of Abeta42. These packing interactions suggest that the protofilament subunits are displaced relative to one another in the Abeta40 and Abeta42 fibril structures. We take advantage of this corrugated structure to design a new class of inhibitors that prevent fibril formation by placing alternating glycine and aromatic residues on one face of a beta-strand. We show that peptide inhibitors based on a GxFxGxF framework disrupt sheet-to-sheet packing and inhibit the formation of mature Abeta fibrils as assayed by thioflavin T fluorescence, electron microscopy, and solid-state NMR spectroscopy. The alternating large and small amino acids in the GxFxGxF sequence are complementary to the corresponding amino acids in the IxGxMxG motif found in the C-terminal sequence of Abeta40 and Abeta42. Importantly, the designed peptide inhibitors significantly reduce the toxicity induced by Abeta42 on cultured rat cortical neurons.     

The role of Phe in the formation of well-ordered oligomers of amyloidogenic hexapeptide (NFGAIL) observed in molecular dynamics simulations with explicit solvent

We observed fast aggregation of partially ordered oligomers in an earlier simulation study of an amyloidogenic hexapeptide NFGAIL. In this work, the nucleation of highly ordered oligomers was further investigated by a combined total of 960 ns molecular dynamics simulations with explicit solvent on NFGAIL and its nonamyloidogenic mutant NAGAIL. In these simulations, four dimer subunits that each was constrained by harmonic forces as a two-strand beta-sheet were used to enhance the rate of formation. It was found that a critical role played by the aromatic residue Phe was to direct the stacking of beta-sheets to form ordered multilayer aggregates. We also found that many molecular arrangements of the peptide satisfied the "cross-beta-structure", a hallmark of amyloid fibrils. The tendency for the peptide to form either parallel or antiparallel beta-sheet was comparable, as was the tendency for the beta-sheets to stack either in parallel or antiparallel orientation. Overall, approximately 85% of the native hexapeptide formed octamers. The fact that only 8% of the octamers were well-ordered species suggests that the dissociation of the disordered oligomers be the rate-limiting step in the formation of highly ordered oligomers. Among the well-ordered subunit pairs, about half was formed by the beta-sheet extension along the main-chain hydrogen-bond direction, whereas the other half was formed by the beta-sheet stacking. Hence, a delicate balance between intersheet and intrasheet interactions appeared to be crucial in the formation of a highly ordered nucleus of amyloid fibrils. The disordered oligomers were mainly stabilized by nonspecific hydrophobic interactions, whereas the well-ordered oligomers were further stabilized by cross-strand hydrogen bonds and favorable side-chain stacking.     

Study of the conformational transition of A beta(1-42) using D-amino acid replacement analogues

A critical event in Alzheimer's disease is the transition of Abeta peptides from their soluble forms into disease-associated beta-sheet-rich conformers. Structural analysis of a complete D-amino acid replacement set of Abeta(1-42) enabled us to localize in the full-length 42-mer peptide the region responsible for the conformational switch into a beta-sheet structure. Although NMR spectroscopy of trifluoroethanol-stabilized monomeric Abeta(1-42) delineated two separated helical domains, only the destabilization of helix I, comprising residues 11-24, caused a transition to a beta-sheet structure. This conformational alpha-to-beta switch was directly accompanied by an aggregation process leading to the formation of amyloid fibrils.     

Beta-sheet breaker peptide inhibitor of Alzheimer's amyloidogenesis with increased blood-brain barrier permeability and resistance to proteolytic degradation in plasma.

Short synthetic peptides homologous to the central region of Abeta but bearing proline residues as beta-sheet blockers have been shown in vitro to bind to Abeta with high affinity, partially inhibit Abeta fibrillogenesis, and redissolve preformed fibrils. While short peptides have been used extensively as therapeutic drugs in medicine, two important problems associated with their use in central nervous system diseases have to be addressed: (a) rapid proteolytic degradation in plasma, and (b) poor blood-brain barrier (BBB) permeability. Recently, we have demonstrated that the covalent modification of proteins with the naturally occurring polyamines significantly increases their permeability at the BBB. We have extended this technology to iAbeta11, an 11-residue beta-sheet breaker peptide that inhibits Abeta fibrillogenesis, by covalently modifying this peptide with the polyamine, putrescine (PUT), and evaluating its plasma pharmacokinetics and BBB permeability. After a single intravenous bolus injection in rats, both 125I-YiAbeta11 and 125I-PUT-YiAbeta11 showed rapid degradation in plasma as determined by trichloroacetic acid (TCA) precipitation and paper chromatography. By switching to the all D-enantiomers of YiAbeta11 and PUT-YiAbeta11, significant protection from degradation by proteases in rat plasma was obtained with only 1.9% and 5.7% degradation at 15 min after intravenous bolus injection, respectively. The permeability coefficient x surface area product at the BBB was five- sevenfold higher in the cortex and hippocampus for the 125I-PUT-D-YiAbeta11 compared to the 125I-D-YiAbeta11, with no significant difference in the residual plasma volume. In vitro assays showed that PUT-D-YiAbeta11 retains its ability to partially inhibit Abeta fibrillogenesis and dissolve preformed amyloid fibrils. Because of its five- to sevenfold increase in permeability at the BBB and its resistance to proteolysis in the plasma, this polyamine-modified beta-sheet breaker peptide may prove to be an effective inhibitor of amyloidogenesis in vivo and, hence, an important therapy for Alzheimer's disease.     

Apoptosis induced in neuronal cells by C-terminal amyloid beta-fragments is correlated with their aggregation properties in phospholipid membranes

A number of findings suggest that lipophilic monomeric Abeta peptides can interact with the cellular lipid membranes. These interactions can affect the membrane integrity and result in the initiation of apoptotic cell death. The secondary structure of C-terminal Abeta peptides (29-40) and the longer (29-42) variant have been investigated in solution by circular dichroism measurements. The secondary structure of lipid bound Abeta (29-40) and (29-42) peptides prepared at different lipid/peptide ratio's, was investigated by ATR-FTIR spectroscopy. Finally, the changes in secondary structure (i.e. the transition of alpha-helix to beta-sheet) of the lipid bound peptides were correlated with the induction of neurotoxic and apoptotic effects in neuronal cells. The data suggest that the C-terminal fragments of the Abeta peptide induce a significant apoptotic cell death, as demonstrated by caspase-3 measurements and DNA laddering, with consistently a stronger effect of the longer Abeta (29-42) variant. Moreover, the induction of apoptotic death induced by these peptides can be correlated with the secondary structure of the lipid bound amyloid beta peptides. Based on these observations, it is proposed that membrane bound aggregated Abeta peptides (produced locally as the result of gamma-secretase cleavage) can accumulate and aggregate in the membrane. These membrane bound beta-sheet aggregated amyloid peptides induce neuronal apoptotic cell death.     

Model of Alzheimer's disease amyloid-beta peptide based on a RNA binding protein

Although Alzheimer's Abeta peptide has been shown to form beta-sheet structure, a high-resolution molecular structure is still unavailable to date. A search for a sequence neighbor using Abeta(10-42) as the query in the Protein Data-Bank (PDB) revealed that an RNA binding protein, AF-Sm1 from Archaeoglobus fulgidus (PDB entry: 1i4k chain Z), shared 36% identical residues. Using AF-Sm1 as a template, we built a molecular model of Abeta(10-42) by applying comparative modeling methods. The model of Abeta(10-42) contains an antiparallel beta-sheet formed by residues 16-23 and 32-41. Hydrophobic surface constituted by residues 17-20 (LVFF) separates distinctly charged regions. Residues that interact with RNA in the AF-Sm1 crystal structure were found to be conserved in Abeta. Using a native gel we demonstrate for the first time that RNA can interact with Abeta and selectively retard the formation of fibrils or higher-order oligomers. We hypothesize that in a similar fashion to AF-Sm1, RNA interacts with Abeta in the beta-hairpin (beta-turn-beta) structure and prevents fibril formation.     

De novo design of a monomeric three-stranded antiparallel beta-sheet

Here we describe the NMR conformational study of a 20-residue linear peptide designed to fold into a monomeric three-stranded antiparallel beta-sheet in aqueous solution. Experimental and statistical data on amino acid beta-turn and beta-sheet propensities, cross-strand side-chain interactions, solubility criteria, and our previous experience with beta-hairpins were considered for a rational selection of the peptide sequence. Sedimentation equilibrium measurements and NMR dilution experiments provide evidence that the peptide is monomeric. Analysis of 1H and 13C-NMR parameters of the peptide, in particular NOEs and chemical shifts, and comparison with data obtained for two 12-residue peptides encompassing the N- and C-segments of the designed sequence indicates that the 20-residue peptide folds into the expected conformation. Assuming a two-state model, the exchange kinetics between the beta-sheet and the unfolded peptide molecules is in a suitable range to estimate the folding rate on the basis of the NMR linewidths of several resonances. The time constant for the coil-beta-sheet transition is of the order of several microseconds in the designed peptide. Future designs based on this peptide system are expected to contribute greatly to our knowledge of the many factors involved in beta-sheet formation and stability.     

Circular dichroism and aggregation studies of amyloid beta (11-8) fragment and its variants

Aggregation of Abeta peptides is a seminal event in Alzheimer's disease. Detailed understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. Here comparative conformational and aggregation studies using CD spectroscopy and thioflavine T fluorescence assay are presented. As a model peptide, the 11-28 fragment of Abeta was used. This model peptide is known to contain the core region responsible for Abeta aggregation. The structural and aggregational behaviour of the peptide was compared with the properties of its variants corresponding to natural, clinically relevant mutants at positions 21-23 (A21G, E22K, E22G, E22Q and D23N). In HFIP (hexafluoro-2-propanol), a strong alpha-helix inducer, the CD spectra revealed an unexpectedly high amount of beta-sheet conformation. The aggregation process of Abeta(11-28) variants provoked by water addition to HFIP was found to be consistent with a model of an alpha-helix-containing intermediate. The aggregation propensity of all Abeta(11-28) variants was also compared and discussed.