Antiparallel Β-sheet Structure Research Articles

The Presence of Antiparallel Beta-Sheets in Toxic Fibrils Formed by ABeta on GM1 Clusters

The abnormal aggregation of amyloid β-protein (Aβ) is considered to be central in the pathogenesis of Alzheimer's disease. We have focused on ‘membrane-mediated’ amyloidogenesis because Yanagisawa et al. identified a specific form of Aβ that was bound to monosialoganglioside GM1 in brains exhibiting the early pathological changes associated with the disease. We have found that amyloid fibrils formed on GM1 clusters were more toxic than those formed in solution [1, 2]. The less toxic fibrils formed in solution are considered to be composed of in-resister parallel β-sheets, whereas the structure of the toxic fibrils is unknown, although FTIR spectra suggested the presence of antiparallel β-sheets [1, 2].In this study, we investigated the structure of the toxic fibrils in detail. Solid-state NMR measurements using site-specifically [15N, 1-13C]-labeled Aβs suggested that the fibrils contained both parallel and antiparallel β-sheet structures. Chemical cross-linking experiments using Cys-substituted Aβs also supported this conclusion. Thus, the toxic fibrils were found to possess a novel unique structure.[1] Fukunaga, S. et al., Biochemistry 51, 8125 (2012).[2] Matsuzaki, K., Acc. Chem. Res. 47, 2397 (2014).

Mechanism of tachyplesin I injury to bacterial membranes and intracellular enzymes, determined by laser confocal scanning microscopy and flow cytometry

Tachyplesin I is a 17 amino acid, cationic, antimicrobial peptide with a typical cyclic antiparallel β-sheet structure. Interactions of tachyplesin I with living bacteria are not well understood, although models have been used to elucidate how tachyplesin I permeabilizes membranes. There are several questions to be answered, such as (i) how does tachyplesin I kill bacteria after it penetrates the membrane and (ii) does bacterial death result from the inactivation of intracellular esterases as well as cell injury? In this study, the dynamic antibacterial processes of tachyplesin I and its interactions with Escherichia coli and Staphylococcus aureus were investigated using laser confocal scanning microscopy in combination with electron microscopy. The effects of tachyplesin I on E. coli cell membrane integrity, intracellular enzyme activity, and cell injury and death were investigated by flow cytometric analysis of cells following single- or double-staining with carboxyfluorescein diacetate or propidium iodide. The results of microscopy indicated that tachyplesin I kills bacteria by acting on the cell membrane and intracellular contents, with the cell membrane representing the primary target. Microscopy results also revealed that tachyplesin I uses different modes of action against E. coli and S. aureus. The results of flow cytometry showed that tachyplesin I caused E. coli cell death mainly by compromising cell membrane integrity and causing the inactivation of intracellular esterases. Flow cytometry also revealed dynamic changes in the different subpopulations of cells with increase in tachyplesin I concentrations. Bacteria exposed to 5μg/mL of tachyplesin I did not die instantaneously; instead, they died gradually via a sublethal injury. However, upon exposure to 10–40μg/mL of tachyplesin I, the bacteria died almost immediately. These results contribute to our understanding of the antibacterial mechanism employed by tachyplesin I.

Formation of lysozyme oligomers at model cell membranes monitored with sum frequency generation spectroscopy.

A growing number of studies suggest that the formation of toxic oligomers, precursors of amyloid fibrils, is initiated at the cell membrane and not in the cytosolic compartments of the cell. Studies of membrane-induced protein oligomerization are challenging due to the difficulties of probing small numbers of proteins present at membrane surfaces. Here, we employ surface-sensitive vibrational sum frequency generation (VSFG) to investigate the secondary structure of lysozyme at the surface of lipid monolayers. We investigate lysozyme aggregation at negatively charged 1,2-dipalmitoyl-sn-glycero-3-(phospho-rac-1-glycerol) (DPPG) lipid monolayers under different pH conditions. The changes in the molecular vibrations of lipids, proteins, and water as a function of pH and surface pressure allow us to simultaneously monitor details of the conformation state of lysozyme, the organization of lipids, and the state of lipid-bound water. At pH = 6 lysozyme induces significant disordering of the lipid layer, and it exists in two states: a monomeric state with a predominantly α-helix content and an oligomeric (za-mer) state. At pH ≤ 3, all membrane-bound lysozyme self-associates into oligomers characterized by an antiparallel β-sheet structure. This is different from the situation in bulk solution, for which circular dichroism (CD) shows that the protein maintains an α-helix conformation, under both neutral and acidic pH conditions. The transition from monomers to oligomers is also associated with a decreased hydration of the lipid monolayer resulting in an increase of the lipid acyl chains ordering. The results indicate that oligomerization requires cooperative action between lysozyme incorporated into the lipid membrane and peripherally adsorbed lysozyme and is associated with the membrane dehydration and lipid reorganization. Membrane-bound oligomers with antiparallel β-sheet structure are found to destabilize lipid membranes.

A comparison of convergently evolved insect silks that share β‐sheet molecular structure

Raspy crickets produce silk webs that are used to build shelters. These webs have been found to consist of both fiber and film components. Raman spectra obtained from both components were found to be very similar for a given species. The protein structure of the fibers and films produced by both species was predominately β-sheet with lesser amounts of β-turns, unordered and α-helical protein also detected. The orientation of the β-sheet backbone in the fiber was determined to be parallel to the fiber axis. Compared to cocoon and dragline silk the orientation distribution exhibits a significant randomly orientated protein component. Amino acid analysis confirmed the presence of glycine, serine, and alanine in both species, which are known to form antiparallel β-sheet structures. Both species, although at significantly different concentrations, where found to contain proline. This amino acid is uncommon in insect silks, and likely involved in increasing fiber elasticity.

Effect of Pulsed Electric Field on Microstructure of Some Amino Acid Group of Soy Protein Isolates

Abstract The effect of a pulsed electric field (PEF) on the microstructure of some amino acids was studied. Raman spectrum was used to determine the effect of PEF on tyrosine, tryptophan, proline residues, histidine, arginine, aliphatic amino acid, disulfide bond, and polypeptide backbone in soy protein isolates (SPI). Results suggested that increasing the intensity of PEF gradually to 50 kV cm−1 led to a reduction in gauche C–S conformation of CCSSCC dihedral angles. The increase of the PEF intensity caused an increase in the gauche–gauche–gauche conformation of the disulfide bond accompanying a decrease in α-helix and β-sheet and an increase in antiparallel β-sheet and disorder structure. A critical pulse intensity of 30 kV cm−1 was observed for unfolding and reassembling of SPI, which was verified in our previous study (Liu et al., Eur Food Res Technol 233:841–50). When the pulse intensity gradually increased to around 30 kV cm−1, the exposure of tyrosine and tryptophan, the vibration of CH2 wagging in proline and CH2 in the midazole ring of histidine, the vibration of C—H bending and C—N stretching inside a charged arginine, and asymmetric H—C—H bending deformation vibration in CH2 and CH3 groups in aromatic and aliphatic amino acids gradually increased, suggesting an unfolding of protein molecules. When the pulse intensity continually increased from 30 to 50 kV cm−1, the microstructure of all above amino acids decreased due to the reassembly of unfolding proteins.

Refined Purification of Large Amounts of Rat cvHsp/HspB7 and Partial Biological Characterization In Vitro

The cardiovascular heat shock protein (cvHsp/HspB7) exhibited cardiac-specific expression and is a possible candidate of dilated cardiomyopathy in heart failure. The molecular characteristics and biochemical properties of cvHsp are only partially understood. This study was aimed to identify the biological properties and molecular high-order structure of cvHsp. The cvHsp protein was prepared by the refined purification at large amount. The pooled fractions were existed as two types of oligomers in solution and exhibited chaperone-like activity. The circular dichroism analyzed ureainduced unfolding processes. Multiple sequence alignment and an automated protein modeling were used to describe the three-dimensional structural model of the cvHsp monomer and dimer. By the refined purification, the cvHsp appeared in oligomeric and dimeric forms (approximately 17 kDa and 40 kDa, respectively) composed of 18.6-kDa monomers. The cvHsp prevented dithiothreitol (DTT)-induced aggregation of the insulin B chain and conferred oligomeric unfolding process in urea-containing solution. It exhibited structural stability and conformed to the two-state folding/unfolding oligomerization model. According to sequence alignment of the rat cvHsp gene, three-dimensional model based on the crystallographic structure of wheat Hsp16.9 was reconstructed. The cvHsp presented two antiparallel β-sheet sandwich structure of sHsp' core α-crystallin domain, and formed dimeric or oligomeric organization in solution. This work described the structural components of cvHsp and existed as the polydispersed molecular oligomers in vitro, which are some common properties of the sHsp family. These characteristics of the cvHsp gene is helpful to clarify molecular functionality in cardiac diseases.

Photophysical study of a π-stacked β-sheet nanofibril forming peptide bolaamphiphile hydrogel

We describe the state of molecular self-assembly of a peptide based bolaamphiphile molecule using spectroscopic and microscopic techniques. The tryptophan and phenylalanine containing peptide bolaamphiphile forms a hydrogel upon sonication under physiological conditions. Sonication helps to reorient the peptide molecules by providing the required energy for the self-assembly process. The disassembly and self-assembly processes are influenced by various stimuli, including heating–cooling and shaking–rest methods. The extensive hydrogen bonding and π–π stacking interactions are responsible for the self-assembly process, which is confirmed by FT-IR, temperature dependent NMR and fluorescence spectroscopy studies. FT-IR and powder X-ray diffraction studies reveal that the gelator molecules self-assemble into an antiparallel β-sheet type structure. The TEM image of the hydrogel shows a well-defined amyloid-like nanofibrillar structure. The amyloid-like behaviour of the fibril forming peptide bolaamphiphile hydrogel is confirmed by ThT and Congo red binding studies. The effect of concentration, time and temperature on the self-assembly mechanism of the peptide bolaamphiphile hydrogel is investigated by time resolved fluorescence spectroscopy.

Solid-State Properties and Vibrational Circular Dichroism Spectroscopy in Solution of Hybrid Foldamers Stereoisomeric Mixtures.

Upon slow evaporation of a 1:1 diastereoisomeric mixture of Boc-(L-Phe-L-Oxd)2 -OBn (1; Boc=tert-butyloxycarbonyl; L-Oxd=trans-(4S,5R)-4-carboxy 5-methyloxazolidin-2-one, Bn=benzyloxycarbonyl) and Boc-L-Phe-L-Oxd-D-Phe-L-Oxd-OBn (2) in methyl tert-butyl ether, single crystals suitable for an X-ray diffraction study were obtained. In contrast, the two pure oligomers lead to the formation of amorphous solids under any crystallization conditions. The preferential conformation of both oligomers was fully elucidated in the solid phase and compared with the known conformation of Boc-(L-Phe-D-Oxd)2 -OBn (3). The preferred conformation of 1 ranges from a polyproline II (PPII) helix to β strands and we can gather that longer and more structured oligomers will form PPII helices. In contrast, compound 3 forms infinite antiparallel β-sheet structures; thus showing the strong effect of the reversal of the absolute configuration of the Oxd moieties on the secondary structure of these hybrid foldamers. The same outcome was retained in solution, as demonstrated by vibrational circular dichroism analysis. Finally, we have demonstrated that a 1:1 mixture of 1 and 2 leads to the formation of new materials with interesting properties that are missing from the two pure compounds, such as the tendency to form crystals, fibers, and globules, depending on the solvent.

Blockade of the formation of insoluble ubiquitinated protein aggregates by EGCG3"Me in the alloxan-induced diabetic kidney.

BackgroundRenal accumulation of reactive carbonyl compounds (RCCs) has been linked to the progression of diabetic nephropathy. We previously demonstrated that carbonyl stress induces the formation of amino-carbonyl cross-links and sharply increases the content of β-sheet-rich structures, which is the seed of insoluble aggregates formation, and tea catechin (-)-epigallocatechin 3-gallate (EGCG) can reverse this process in vitro and in vivo. In this study, methylated derivative (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3”Me) was hypothesized to neutralize carbonyl stress mediating the formation of insoluble ubiquitinated protein (IUP) aggregates, and reduce the early development of diabetic nephropathy.Methods and resultsDiabetes was induced in mice by intraperitoneally injecting alloxan monohydrate (200 mg/kg/d) twice and administering EGCG3”Me by gavage for 15 d. Reagent case and western blot results showed that, in diabetic kidneys, the carbonyl proteins in the serum increased; and in insoluble protein fraction, 4-hydroxynonenal-modified proteins, IUP aggregates and p62 accumulated; FT-IR study demonstrated that the lipid content, anti-parallel β-sheet structure and aggregates increased. EGCG3”Me treatment could effectively reverse this process, even better than the negative control treatment.ConclusionsEGCG3”Me exhibiting anti-β-sheet-rich IUP aggregate properties, maybe represents a new strategy to impede the progression of diabetic nephropathy and other diabetic complications.

Molecular Structure of RADA16-I Designer Self-Assembling Peptide Nanofibers

The designer self-assembling peptide RADA16-I forms nanofiber matrices which have shown great promise for regenerative medicine and three-dimensional cell culture. The RADA16-I amino acid sequence has a β-strand-promoting alternating hydrophobic/charged motif, but arrangement of β-strands into the nanofiber structure has not been previously determined. Here we present a structural model of RADA16-I nanofibers, based on solid-state NMR measurements on samples with different schemes for (13)C isotopic labeling. NMR peak positions and line widths indicate an ordered structure composed of β-strands. The NMR data show that the nanofibers are composed of two stacked β-sheets stabilized by a hydrophobic core formed by alanine side chains, consistent with previous proposals. However, the previously proposed antiparallel β-sheet structure is ruled out by measured (13)C-(13)C dipolar couplings. Instead, neighboring β-strands within β-sheets are parallel, with a registry shift that allows cross-strand staggering of oppositely charged arginine and aspartate side chains. The resulting structural model is compared to nanofiber dimensions observed via images taken by transmission electron microscopy and atomic force microscopy. Multiple NMR peaks for each alanine side chain were observed and could be attributed to multiple configurations of side chain packing within a single scheme for intermolecular packing.

C–H Stretch for Probing Kinetics of Self-Assembly into Macromolecular Chiral Structures at Interfaces by Chiral Sum Frequency Generation Spectroscopy

Self-assembly of molecules into chiral macromolecular and supramolecular structures at interfaces is important in various fields, such as biomedicine, polymer sciences, material sciences, and supramolecular chemistry. However, probing the kinetics at interfaces remains challenging because it requires a real-time method that has selectivity to both interface and chirality. Here, we introduce an in situ approach of using the C-H stretch as a vibrational probe detected by chiral sum frequency generation spectroscopy (cSFG). We showed that the C-H stretch cSFG signals of an amphiphilic peptide (LK7β) can reveal the kinetics of its self-assembly into chiral β-sheet structures at the air-water interface. The cSFG experiments in conjunction with measurements of surface pressure allow us to propose a mechanism of the self-assembly process, which involves an immediate adsorption of disordered structures followed by a lag phase before the self-assembly into chiral antiparallel β-sheet structures. Our method of using the C-H stretch signals implies a general application of cSFG to study the self-assembly of bioactive, simple organic, and polymeric molecules into chiral macromolecular and supramolecular structures at interfaces, which will be useful in tackling problems, such as protein aggregation, rational design of functional materials, and fabrication of molecular devices.

Induction of Anti-influenza Immunity by Modified Green Fluorescent Protein (GFP) Carrying Hemagglutinin-derived Epitope Structure

The development of vaccination methods that can overcome the emergence of new types of influenza strains caused by escape mutations is desirable to avoid future pandemics. Here, a novel type of immunogen was designed that targeted the conformation of a highly conserved region of influenza A virus hemagglutinin (HA) composed of two separate sequences that associate to form an anti-parallel β-sheet structure. Our previous study identified this β-sheet region as the structural core in the epitope of a characteristic antibody (B-1) that strongly neutralizes a wide variety of strains within the H3N2 serotype, and therefore this β-sheet region was considered a good target to induce broadly reactive immunity against the influenza A virus. To design the immunogen, residues derived from the B-1 epitope were introduced directly onto a part of enhanced green fluorescent protein (EGFP), whose surface is mostly composed of β-sheets. Through site-directed mutagenesis, several modified EGFPs with an epitope-mimicking structure embedded in their surface were prepared. Two EGFP variants, differing from wild-type (parental) EGFP by only five and nine residues, induced mice to produce antibodies that specifically bind to H3-type HA and neutralize H3N2 virus. Moreover, three of five mice immunized with each of these EGFP variants followed by a booster with equivalent mCherry variants acquired anti-viral immunity against challenge with H3N2 virus at a lethal dosage. In contrast to conventional methods, such as split HA vaccine, preparation of this type of immunogen requires less time and is therefore expected to be quickly responsive to newly emerged influenza viral strains.

Molecular Structures of Amyloid and Prion Fibrils: Consensus versus Controversy

Many peptides and proteins self-assemble into amyloid fibrils. Examples include mammalian and fungal prion proteins, polypeptides associated with human amyloid diseases, and proteins that may have biologically functional amyloid states. To understand the propensity for polypeptides to form amyloid fibrils and to facilitate rational design of amyloid inhibitors and imaging agents, it is necessary to elucidate the molecular structures of these fibrils. Although fibril structures were largely mysterious 15 years ago, a considerable body of reliable structural information about amyloid fibril structures now exists, with essential contributions from solid state nuclear magnetic resonance (NMR) measurements. This Account reviews results from our laboratories and discusses several structural issues that have been controversial. In many cases, the amino acid sequences of amyloid fibrils do not uniquely determine their molecular structures. Self-propagating, molecular-level polymorphism complicates the structure determination problem and can lead to apparent disagreements between results from different laboratories, particularly when different laboratories study different polymorphs. For 40-residue β-amyloid (Aβ₁₋₄₀) fibrils associated with Alzheimer's disease, we have developed detailed structural models from solid state NMR and electron microscopy data for two polymorphs. These polymorphs have similar peptide conformations, identical in-register parallel β-sheet organizations, but different overall symmetry. Other polymorphs have also been partially characterized by solid state NMR and appear to have similar structures. In contrast, cryo-electron microscopy studies that use significantly different fibril growth conditions have identified structures that appear (at low resolution) to be different from those examined by solid state NMR. Based on solid state NMR and electron paramagnetic resonance (EPR) measurements, the in-register parallel β-sheet organization found in β-amyloid fibrils also occurs in many other fibril-forming systems. We attribute this common structural motif to the stabilization of amyloid structures by intermolecular interactions among like amino acids, including hydrophobic interactions and polar zippers. Surprisingly, we have recently identified and characterized antiparallel β-sheets in certain fibrils that are formed by the D23N mutant of Aβ₁₋₄₀, a mutant that is associated with early-onset, familial neurodegenerative disease. Antiparallel D23N-Aβ₁₋₄₀ fibrils are metastable with respect to parallel structures and, therefore, represent an off-pathway intermediate in the amyloid fibril formation process. Other methods have recently produced additional evidence for antiparallel β-sheets in other amyloid-formation intermediates. As an alternative to simple parallel and antiparallel β-sheet structures, researchers have proposed β-helical structural models for some fibrils, especially those formed by mammalian and fungal prion proteins. Solid state NMR and EPR data show that fibrils formed in vitro by recombinant PrP have in-register parallel β-sheet structures. However, the structure of infectious PrP aggregates is not yet known. The fungal HET-s prion protein has been shown to contain a β-helical structure. However, all yeast prions studied by solid state NMR (Sup35p, Ure2p, and Rnq1p) have in-register parallel β-sheet structures, with their Gln- and Asn-rich N-terminal segments forming the fibril core.

Study of the aggregation mechanism of polyglutamine peptides using replica exchange molecular dynamics simulations

Polyglutamine (polyQ, a peptide) with an abnormal repeat length is the causative agent of polyQ diseases, such as Huntington's disease. Although glutamine is a polar residue, polyQ peptides form insoluble aggregates in water, and the mechanism for this aggregation is still unclear. To elucidate the detailed mechanism for the nucleation and aggregation of polyQ peptides, replica exchange molecular dynamics simulations were performed for monomers and dimers of polyQ peptides with several chain lengths. Furthermore, to determine how the aggregation mechanism of polyQ differs from those of other peptides, we compared the results for polyQ with those of polyasparagine and polyleucine. The energy barrier between the monomeric and dimeric states of polyQ was found to be relatively low, and it was observed that polyQ dimers strongly favor the formation of antiparallel β-sheet structures. We also found a characteristic behavior of the monomeric polyQ peptide: a turn at the eighth residue is always present, even when the chain length is varied. We previously showed that a structure including more than two sets of β-turns is stable, so a long monomeric polyQ chain can act as an aggregation nucleus by forming several pairs of antiparallel β-sheet structures within a single chain. Since the aggregation of polyQ peptides has some features in common with an amyloid fibril, our results shed light on the mechanism for the aggregation of polyQ peptides as well as the mechanism for the formation of general amyloid fibrils, which cause the onset of amyloid diseases.

Modulation of amyloid-β 1-42 structure and toxicity by proline-rich whey peptides

A proline-rich peptide product prepared from bovine whey protein that was enriched in several hydrophobic amino acids including proline (whey proline-rich peptide, wPRP) was shown to modulate the folding pathway of human amyloid beta peptide 1-42 (Aβ42) into oligomers. Concentration-dependent changes in ThT-binding to Ab42 by wPRP indicated suppression of oligomerisation, that was supported by Transmission Electron Microscopy. Suppression of β-sheet and specifically, anti-parallel β-sheet structures by wPRP was demonstrated by ATR-FTIR spectroscopy, where evidence for capacity of wPRP to dissociate pre-existing β-sheet structures in Aβ42 was also apparent. Suppression of anti-parallel β-sheets of oligomeric Aβ42 was associated with rescue of yeast and SH-SY5Y neuronal cells providing important evidence for the association between anti-parallel β-sheet structure and oligomer toxicity. It was proposed that the interaction of wPRP with Aβ42 interfered with the anti-parallel folding pathway of oligomeric Aβ42 and ultimately produced 'off-pathway' structures of lowered total β-sheet content, with attenuated cellular toxicity.

Immunization of rabbits with synthetic peptides derived from a highly conserved β-sheet epitope region underneath the receptor binding site of influenza A virus

BackgroundThere is increasing concern about the speed with which health care providers can administer prophylaxis and treatment in an influenza pandemic. Generally, it takes several months to manufacture an influenza vaccine by propagation of the virus in chicken eggs or cultured cells. Newer, faster protocols for the production of vaccines that induce broad-spectrum immunity are therefore highly desirable. We previously developed human monoclonal antibody B-1 that shows broadly neutralizing activity against influenza A virus H3N2. B-1 recognizes an epitope region that includes an antiparallel β-sheet structure underneath the receptor binding site of influenza hemagglutinin (HA). In this study, the efficacy of a synthetic peptide vaccine derived from this epitope region against influenza A was evaluated.Materials and methodsTwo peptides were synthesized, the upper and lower peptides. These peptides comprise amino acid residues 167–187 and 225–241, respectively, of the B-1 epitope region of HA, which is involved in forming the β-sheet structure. Both peptides were then coupled to keyhole limpet hemocyanin, and the peptides, alone or in combination, were used to immunize rabbits. The resulting antibody responses were examined by enzyme-linked immunosorbent assay. The upper peptide, but not the lower peptide, elicited antibodies that were reactive to HA. Interestingly, the use of both peptides together could elicit antibodies with a higher reactivity to HA than either peptide alone. The antibodies were found to react to HA at the N-terminus of the upper peptide, which is exposed at the surface of trimeric HA on influenza virions.DiscussionThe higher production of HA-reactive antibodies following immunization with both peptides suggests that the upper peptide forms the effective epitope structure in the binding state, and the lower peptide enhances the production of HA antibodies. This study could be the first step towards the development of pandemic viral vaccines that can be produced within short time periods.

Insight into Sup35(7-13) Misfolding and Interactions using Molecular Dynamics Simulation

The misfolding of amyloidogenic proteins as a molecular switch can lead to Alzheimer's, Parkinson's, prion diseases and other neurodegenerative diseases. However, the molecular mechanism of the misfolding is not well understood. In our recent AFM force spectroscopy study of a yeast prion Sup35 protein fragment (CGNNQQNY), we have shown that this peptide forms stable misfolded dimers. Electrostatic interactions contribute to the formation of dimers and peptide self-assembly into aggregates1. In this study, we applied Molecular Dynamics (MD) simulation to elucidate the mechanism by which the peptide assembles into dimers. At μs timescale, the isolated peptide monomer [Cys6]Sup35(6-13) adopted a non-structured β-turn/bend-like conformations. Upon approaching, the two monomers changed their initial conformation and formed a dimer with a well-defined antiparallel β-sheet structure. Steered MD approach was applied to analyze the rupture process for the dimer. The results showed that the dimer dissociates cooperatively resulting in a sharp rupture peak corresponding to breakage of the β-sheet structure. Altogether, the MD simulations revealed a critical role of the interpeptide interaction in the misfolding process, and provided additional support to our hypothesis on the key role of the dimerization on the amyloid aggregation process.

NMR Analysis of Cross Strand Aromatic Interactions in an 8 Residue Hairpin and a 14 Residue Three Stranded β-Sheet Peptide

Cross strand aromatic interactions between a facing pair of phenylalanine residues in antiparallel β-sheet structures have been probed using two structurally defined model peptides. The octapeptide Boc-LFV(D)P(L)PLFV-OMe (peptide 1) favors the β-hairpin conformation nucleated by the type II' β-turn formed by the (D)Pro-(L)Pro segment, placing Phe2 and Phe7 side chains in proximity. Two centrally positioned (D)Pro-(L)Pro segments facilitate the three stranded β-sheet formation in the 14 residue peptide Boc-LFV(D)P(L)PLFVA(D)P(L)PLFV-OMe (peptide 2) in which the Phe2/Phe7 orientations are similar to that in the octapeptide. The anticipated folded conformations of peptides 1 and 2 are established by the delineation of intramolecularly hydrogen bonded NH groups and by the observation of specific cross strand NOEs. The observation of ring current shifted aromatic protons is a diagnostic of close approach of the Phe2 and Phe7 side chains. Specific assignment of aromatic proton resonances using HSQC and HSQC-TOCSY methods allow an analysis of interproton NOEs between the spatially proximate aromatic rings. This approach facilitates specific assignments in systems containing multiple aromatic rings in spectra at natural abundance. Evidence is presented for a dynamic process which invokes a correlated conformational change about the C(α)-C(β)(χ(1)) bond for the pair of interacting Phe residues. NMR results suggest that aromatic ring orientations observed in crystals are maintained in solution. Anomalous temperature dependence of ring current induced proton chemical shifts suggests that solvophobic effects may facilitate aromatic ring clustering in apolar solvents.

GM1 Cluster Mediates Formation of Toxic Aβ Fibrils by Providing Hydrophobic Environments

The conversion of soluble, nontoxic amyloid β-proteins (Aβ) to aggregated, toxic forms rich in β-sheets is considered to be a key step in the development of Alzheimer's disease. Accumulating evidence suggests that lipid-protein interactions play a crucial role in the aggregation of amyloidogenic proteins like Aβ. Our group has previously reported that amyloid fibrils of Aβ formed on membranes containing clusters of GM1 ganglioside (M-fibrils) exhibit greater cytotoxicity than fibrils formed in aqueous solution (W-fibrils) [ Okada ( 2008 ) J. Mol. Biol. 382 , 1066 - 1074 ]. W-fibrils are considered to consist of in-register parallel β-sheets. However, the precise molecular structure of M-fibrils and force driving the formation of toxic fibrils remain unclear. In this study, we hypothesized that low-polarity environments provided by GM1 clusters drive the formation of toxic fibrils and compared the structure and cytotoxicity of W-fibrils, M-fibrils, and aggregates formed in a low-polarity solution mimicking membrane environments. First, we determined solvent conditions which mimic the polarity of raftlike membranes using Aβ-(1-40) labeled with the 7-diethylaminocoumarin-3-carbonyl dye. The polarity of a mixture of 80% 1,4-dioxane and 20% water (v/v) was found to be close to that of raftlike membranes. Aβ-(1-40) formed amyloid fibrils within several hours in 80% dioxane (D-fibrils) or in the presence of raftlike membranes, whereas a much longer incubation time was required for fibril formation in a conventional buffer. D-fibrils were morphologically similar to M-fibrils. Fourier-transform infrared spectroscopy suggested that M-fibrils and D-fibrils contained antiparallel β-sheets. These fibrils had greater surface hydrophobicity and exhibited significant toxicity against human neuroblastoma SH-SY5Y cells, whereas W-fibrils with less surface hydrophobicity were not cytotoxic. We concluded that ganglioside clusters mediate the formation of toxic amyloid fibrils of Aβ with an antiparallel β-sheet structure by providing less polar environments.

Quercetin protects against cadmium-induced biochemical and structural changes in rat liver revealed by FT-IR spectroscopy

Cadmium (Cd) is a toxic metal occurring in the environment naturally and as a pollutant emanating from industrial and agricultural sources. Quercetin is a naturally occurring flavonoid which has been reported to have a wide range of pharmacological properties. The aim of the present study is to investigate the protective effect of quercetin in modifying the biochemical and structural changes at the molecular level in Cd-intoxicated liver tissues in Wistar rats by using Fourier transform infrared (FT-IR) spectroscopy. FT-IR spectroscopy allows us rapid and sensitive determination of functional groups belonging to proteins, lipids, carbohydrates and nucleic acids simultaneously. The results revealed that there is a significant decrease in the amount of unsaturated lipids in Cd-intoxicated liver, which indicates an increase in lipid peroxidation. In addition, there is a significant increase in the amount of saturated lipids, collagen and glycogen contents in the Cd-intoxicated liver tissues. Moreover, the significant increase in lipid order and a decrease in protein to lipid ratio are observed. Furthermore, a decrease in the intensity/area of the amide I band and shifting of the position of this band to lower frequency values in Cd-intoxicated liver indicates structural changes in proteins. Detailed secondary structure analysis of the amide I band reveals a significant increase in random coil and antiparallel β-sheet structures and a decrease in α-helix and aggregated β-sheet structures, which indicates protein denaturation due to oxidative modification. Administration of quercetin significantly decreases the levels of hepatic lipids and collagen that are found to increase in Cd-intoxicated liver tissues. In conclusion, the results of the present study suggest that quercetin may exert its antioxidative effect by modulating the biochemical and structural changes at the molecular level during Cd-intoxicated liver tissues, which can be detected by using FT-IR spectroscopic technique.