Computational design and evaluation of β-sheet breaker peptides for destabilizing Alzheimer's amyloid-β42 protofibrils

The β-sheet breaker (BSB) peptides interfere with amyloid fibril assembly and used as therapeutic agents in the treatment of Alzheimer's disease (AD). In this regard, a simple yet effective in silico screening methodology was applied in the present study to evaluate a potential 867 pentapeptide library based on known BSB peptide, LPFFD, for destabilizing Aβ₄₂ protofibrils. The molecular docking based virtual screening was used to filter out pentapeptides having binding affinities stronger than LPFFD. In the next step, binding free energies of the top 10 pentapeptides were evaluated using the MM-PBSA method. The residue-wise binding free energy analysis reveals that two pentapeptides, PVFFE, and PPFYE, bind to the surface of Aβ₄₂ protofibril and another pentapeptide, PPFFE, bind in the core region of Aβ₄₂ protofibril. By employing molecular dynamics simulation as a post filter for the top-hit peptides from MM-PBSA, the pentapeptides, PPFFE, PVFFE, and PPFYE, have been identified as potential BSB peptides for destabilizing Aβ₄₂ protofibril structure. The conformational microstate analysis, a significant decrease in the β-sheet content of Aβ₄₂ protofibril, a loss in the total number of hydrogen bonds in Aβ₄₂ protofibril, Asp23-Lys28 salt bridge destabilization and analysis of the free energy surfaces highlight Aβ₄₂ protofibril structure destabilization in presence of pentapeptides. Among three top-hit pentapeptides, PPFFE displayed the most potent Aβ₄₂ protofibril destabilization effect that shifted the energy minima toward lowest value of β-sheet content as well as lowest number of hydrogen bonds in Aβ₄₂ protofibril. The in silico screening workflow presented in the study highlight an alternative tool for designing novel peptides with enhanced BSB ability as potential therapeutic agents for AD.     

Insights into the inhibitory mechanism of a resveratrol and clioquinol hybrid against Aβ42 aggregation and protofibril destabilization: A molecular dynamics simulation study

Amyloid-β (Aβ) peptide instinctively aggregate and form plaques in the brain of Alzheimer’s disease (AD) patients. At present, there is no cure or treatment for AD, and significant effort has, therefore, been made to discover potent drugs against AD. Previous studies reported that a resveratrol and clioquinol hybrid compound [(E)-5-(4-hydroxystyryl)quinolone-8-ol], C1, strongly inhibit Aβ₄₂ aggregation and disassemble preformed fibrils. However, the atomic level details of the inhibitory mechanism of C1 against Aβ₄₂ aggregation and protrofibril disassembly remains elusive. In this regard, molecular docking and molecular dynamics (MD) simulation of Aβ₄₂ monomer, Aβ₄₂ monomer–C1 complex, Aβ₄₂ protofibril, and Aβ₄₂ protofibril–C1 complex were performed in the present study. MD simulations highlighted that C1 bind in the central hydrophobic core (CHC) region, i.e., KLVFF (16–20) of Aβ₄₂ monomer, which plays a critical role in Aβ₄₂ aggregation. C1 promote the formation of native helical conformation in the Aβ₄₂ monomer and decrease the probability of D23–K28 salt bridge interaction that is critical in the formation of aggregation-prone β-sheet conformation. Further, C1 destabilize Aβ₄₂ protofibril structure by increasing the interchain distance between chains A–B, disrupting the salt–bridge interaction between D23–K28, and decreasing the number of backbone hydrogen bonds between chains A–B of the Aβ₄₂ protofibril structure. The insights into the underlying inhibitory mechanism of small molecules that display potential in vitro anti–aggregation activity against Aβ₄₂ will be beneficial for the rational design of more potent drug molecules against AD.

Communicated by Ramaswamy H. Sarma     

Fe2+ binding on amyloid β‐peptide promotes aggregation

The metal ions Zn²⁺, Cu²⁺, and Fe²⁺ play a significant role in the aggregation mechanism of Aβ peptides. However, the nature of binding between metal and peptide has remained elusive; the detailed information on this from the experimental study is very difficult. Density functional theory (dft) (M06‐2X/6‐311++G (2df,2pd) +LANL2DZ) has employed to determine the force field resulting due to metal and histidine interaction. We performed 200 ns molecular dynamics (MD) simulation on Aβ_1‐42‐Zn²⁺, Aβ_1‐42‐Cu²⁺, and Aβ_1‐42‐Fe²⁺ systems in explicit water with different combination of coordinating residues including the three Histidine residues in the N‐terminal. The present investigation, the Aβ_1‐42‐Zn²⁺ system possess three turn conformations separated by coil structure. Zn²⁺ binding caused the loss of the helical structure of N‐terminal residues which transformed into the S‐shaped conformation. Zn²⁺ has reduced the coil and increases the turn content of the peptide compared with experimental study. On the other hand, the Cu²⁺ binds with peptide, β sheet formation is observed at the N‐terminal residues of the peptide. Fe²⁺ binding is to promote the formation of Glu22‐Lys28 salt‐bridge which stabilized the turn conformation in the Phe19‐Gly25 residues, subsequently β sheets were observed at His13‐Lys18 and Gly29‐Gly37 residues. The turn conformation facilitates the β sheets are arranged in parallel by enhancing the hydrophobic contact between Gly25 and Met35, Lys16 and Met35, Leu17 and Leu34, Val18 and Leu34 residues. The Fe²⁺ binding reduced the helix structure and increases the β sheet content in the peptide, which suggested, Fe²⁺ promotes the oligomerization by enhancing the peptide‐peptide interaction. Proteins 2016; 84:1257–1274. © 2016 Wiley Periodicals, Inc.     

Scrutiny of the mechanism of small molecule inhibitor preventing conformational transition of amyloid-β42 monomer: insights from molecular dynamics simulations

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is characterized by loss of intellectual functioning of brain and memory loss. According to amyloid cascade hypothesis, aggregation of amyloid-β₄₂ (Aβ₄₂) peptide can generate toxic oligomers and their accumulation in the brain is responsible for the onset of AD. In spite of carrying out a large number of experimental studies on inhibition of Aβ₄₂ aggregation by small molecules, the detailed inhibitory mechanism remains elusive. In the present study, comparable molecular dynamics (MD) simulations were performed to elucidate the inhibitory mechanism of a sulfonamide inhibitor C1 (2,5-dichloro-N-(4-piperidinophenyl)-3-thiophenesulfonamide), reported for its in vitro and in vivo anti-aggregation activity against Aβ₄₂. MD simulations reveal that C1 stabilizes native α-helix conformation of Aβ₄₂ by interacting with key residues in the central helix region (13–26) with hydrogen bonds and π–π interactions. C1 lowers the solvent-accessible surface area of the central hydrophobic core (CHC), KLVFF (16–20), that confirms burial of hydrophobic residues leading to the dominance of helical conformation in the CHC region. The binding free energy analysis with MM–PBSA demonstrates that Ala2, Phe4, Tyr10, Gln15, Lys16, Leu17, Val18, Phe19, Phe20, Glu22, and Met35 contribute maximum to binding free energy (?43.1 kcal/mol) between C1 and Aβ₄₂ monomer. Overall, MD simulations reveal that C1 inhibits Aβ₄₂ aggregation by stabilizing native helical conformation and inhibiting the formation of aggregation-prone β-sheet conformation. The present results will shed light on the underlying inhibitory mechanism of small molecules that show potential in vitro anti-aggregation activity against Aβ₄₂.     

De novo design,synthesis and solution conformational study of two didehydroundecapeptides: effect of nature and number of amino acids interspersed between Phe residues

De novo design of peptides and proteins has recently surfaced as an approach for investigating protein structure and function. This approach vitally tests our knowledge of protein folding and function, while also laying the groundwork for the fabrication of proteins with properties not precedented in nature. The success relies heavily on the ability to design relatively short peptides that can espouse stable secondary structures. To this end, substitution with α,β‐didehydroamino acids, especially α,β‐didehydrophenylalanine (Δ^zPhe), comes in use for spawning well‐defined structural motifs. Introduction of ΔPhe induces β‐bends in small and 3₁₀‐helices in longer peptide sequences. The present work aims to investigate the effect of nature and the number of amino acids interspersed between two ΔPhe residues in two model undecapeptides, Ac‐Gly‐Ala‐ΔPhe‐Ile‐Val‐ΔPhe‐Ile‐Val‐ΔPhe‐Ala‐Gly‐NH₂ (I) and Boc‐Val‐ΔPhe‐Phe‐Ala‐Phe‐ΔPhe‐Phe‐Leu‐Ala‐ΔPhe‐Gly‐OMe (II). Peptide I was synthesized using solid‐phase chemistry and characterized using circular dichroism spectroscopy. Peptide II was synthesized using solution‐phase chemistry and characterized using circular dichroism and nuclear magnetic resonance spectroscopy. Peptide I was designed to examine the effect of incorporating β‐strand‐favoring residues like valine and isoleucine as spacers between two ΔPhe residues on the final conformation of the resulting peptide. Circular dichroism studies on this peptide have shown the existence of a 3₁₀‐helical conformation. Peptide II possesses three amino acids as spacers between ΔPhe residues and has been reported to adopt a mixed 3₁₀/α‐helical conformation using circular dichroism and nuclear magnetic resonance spectroscopy studies. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Dimerization effects on coacervation property of an elastin‐derived synthetic peptide (FPGVG)5

Elastin, a core protein of the elastic fibers, exhibits the coacervation (temperature‐dependent reversible association/dissociation) under physiological conditions. Because of this characteristic, elastin and elastin‐derived peptides have been considered to be useful as base materials for developing various biomedical products, skin substitutes, synthetic vascular grafts, and drug delivery systems. Although elastin‐derived polypeptide (Val‐Pro‐Gly‐Val‐Gly)_n also has been known to demonstrate coacervation property, a sufficiently high (VPGVG)_n repetition number (n > 40) is required for coacervation. In the present study, a series of elastin‐derived peptide (Phe‐Pro‐Gly‐Val‐Gly)₅ dimers possessing high coacervation potential were newly developed. These novel dimeric peptides exhibited coacervation at significantly lower concentrations and temperatures than the commonly used elastin‐derived peptide analogs; this result suggests that the coacervation ability of the peptides is enhanced by dimerization. Circular dichroism (CD) measurements indicate that the dimers undergo similar temperature‐dependent and reversible conformational changes when coacervation occurs. The molecular dynamics calculation results reveal that the sheet‐turn‐sheet motif involving a type II β‐turn‐like structure commonly observed among the dimers and caused formation of globular conformation of them. These synthesized peptide dimers may be useful not only as model peptides for structural analysis of elastin and elastin‐derived peptides, but also as base materials for developing various temperature‐sensitive biomedical and industrial products. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Dual effects of familial Alzheimer's disease mutations (D7H,D7N,and H6R) on amyloid β peptide: Correlation dynamics and zinc binding

Although the N‐terminal region of Amyloid β (Aβ) peptides plays dual roles as metal‐coordinating sites and conformational modulator, few studies have been performed to explore the effects of mutations at this region on the overall conformational ensemble of Aβ and the binding propensity of metal ions. In this work, we focus on how three familial Alzheimer's disease mutations (D7H, D7N, and H6R) alter the structural characteristics and thermodynamic stabilities of Aβ42 using molecular dynamics simulations. We observe that each mutation displays increased β‐sheet structures in both N and C termini. In particular, both the N terminus and central hydrophobic region of D7H can form stable β‐hairpin structures with its C terminus. The conserved turn structure at Val²⁴–Lys²⁸ in all peptides and Zn²⁺‐bound Aβ42 is confirmed as the common structural motif to nucleate folding of Aβ. Each mutant can significantly increase the solvation free energy and thus enhance the aggregation of Aβ monomers. The correlation dynamics between Aβ(1–16) and Aβ(17–42) fragments are elucidated by linking the domain motions with the corresponding structured conformations. We characterize the different populations of correlated domain motions for each mutant from a more macroscopic perspective, and unexpectedly find that Zn²⁺‐bound Aβ42 ensemble shares the same populations as Aβ42, indicating that the binding of Zn²⁺ to Aβ follows the conformational selection mechanism, and thus is independent of domain motions, even though the structures of Aβ have been modified at a residue level. Proteins 2014; 82:3286–3297. © 2014 Wiley Periodicals, Inc.     

Elucidating the role of Trp105 in the KPC-2 ��-lactamase

The molecular basis of resistance to β‐lactams and β‐lactam‐β‐lactamase inhibitor combinations in the KPC family of class A enzymes is of extreme importance to the future design of effective β‐lactam therapy. Recent crystal structures of KPC‐2 and other class A β‐lactamases suggest that Ambler position Trp105 may be of importance in binding β‐lactam compounds. Based on this notion, we explored the role of residue Trp105 in KPC‐2 by conducting site‐saturation mutagenesis at this position. Escherichia coli DH10B cells expressing the Trp105Phe, ‐Tyr, ‐Asn, and ‐His KPC‐2 variants possessed minimal inhibitory concentrations (MICs) similar to E. coli cells expressing wild type (WT) KPC‐2. Interestingly, most of the variants showed increased MICs to ampicillin‐clavulanic acid but not to ampicillin‐sulbactam or piperacillin‐tazobactam. To explain the biochemical basis of this behavior, four variants (Trp105Phe, ‐Asn, ‐Leu, and ‐Val) were studied in detail. Consistent with the MIC data, the Trp105Phe β‐lactamase displayed improved catalytic efficiencies, k_cat/K_m, toward piperacillin, cephalothin, and nitrocefin, but slightly decreased k_cat/K_m toward cefotaxime and imipenem when compared to WT β‐lactamase. The Trp105Asn variant exhibited increased K_ms for all substrates. In contrast, the Trp105Leu and ‐Val substituted enzymes demonstrated notably decreased catalytic efficiencies (k_cat/K_m) for all substrates. With respect to clavulanic acid, the K_is and partition ratios were increased for the Trp105Phe, ‐Asn, and ‐Val variants. We conclude that interactions between Trp105 of KPC‐2 and the β‐lactam are essential for hydrolysis of substrates. Taken together, kinetic and molecular modeling studies define the role of Trp105 in β‐lactam and β‐lactamase inhibitor discrimination.     

Molecular mechanism of β-sheet self-organization at water-hydrophobic interfaces

The capacity to form β‐sheet structure and to self‐organize into amyloid aggregates is a property shared by many proteins. Severe neurodegenerative pathologies such as Alzheimer's disease are thought to involve the interaction of amyloidogenic protein oligomers with neuronal membranes. To understand the experimentally observed catalysis of amyloid formation by lipid membranes and other water‐hydrophobic interfaces, we examine the physico‐chemical basis of peptide adsorption and aggregation in a model membrane using atomistic molecular simulations. Blocked octapeptides with simple, repetitive sequences, (Gly‐Ala)₄, and (Gly‐Val)₄, are used as models of β‐sheet‐forming polypeptide chains found in the core of amyloid fibrils. In the presence of an n‐octane phase mimicking the core of lipid membranes, the peptides spontaneously partition at the octane‐water interface. The adsorption of nonpolar sidechains displaces the peptides' conformational equilibrium from a heterogeneous ensemble characterized by a high degree of structural disorder toward a more ordered ensemble favoring β‐hairpins and elongated β‐strands. At the interface, peptides spontaneously aggregate and rapidly evolve β‐sheet structure on a 10 to 100 ns time scale, while aqueous aggregates remain amorphous. Catalysis of β‐sheet formation results from the combination of the hydrophobic effect and of reduced conformational entropy of the polypeptide chain. While the former drives interfacial partition and displaces the conformational equilibrium of monomeric peptides, the planar interface further facilitates β‐sheet organization by increasing peptide concentration and reducing the dimensionality of self‐assembly from three to two. These findings suggest a general mechanism for the formation of β‐sheets on the surface of globular proteins and for amyloid self‐organization at hydrophobic interfaces. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Aromatic‐interaction‐mediated inhibition of β‐amyloid assembly structures and cytotoxicity

Abnormal aggregation of β‐amyloid (Aβ) peptide plays an important role in the onset and progress of Alzheimer's disease (AD); hence, targeting Aβ aggregation is considered as an effective therapeutic strategy. Here, we studied the aromatic‐interaction‐mediated inhibitory effect of oligomeric polypeptides (K8Y8, K4Y8, K8W8) on Aβ42 fibrillization process. The polypeptides containing lysine as well as representative aromatic amino acids of tryptophan or tyrosine were found to greatly suppress the aggregation as evaluated by thioflavin T assay. Circular dichroism spectra showed that the β‐sheet formation of Aβ42 peptides decreased with the polypeptide additives. Molecular docking studies revealed that the oligomeric polypeptides could preferentially bind to Aβ42 through π–π stacking between aromatic amino acids and Phe19, together with hydrogen bonding. The cell viability assay confirmed that the toxicity of Aβ42 to SH‐SY5Y cells was markedly reduced in the presence of polypeptides. This study could be beneficial for developing peptide‐based inhibitory agents for amyloidoses. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Multi-target-directed triazole derivatives as promising agents for the treatment of Alzheimer’s disease

A novel series of triazole-based compounds have been designed, synthesised and evaluated as multi-target-directed ligands (MTDLs) against Alzheimer disease (AD). The triazole-based compounds have been designed to target four major AD hallmarks that include Aβ aggregation, metal-induced Aβ aggregation, metal dys-homeostasis and oxidative stress. Among the synthesised compounds, 6n having o-CF₃ group on the phenyl ring displayed most potent inhibitory activity (96.89% inhibition, IC₅₀ = 8.065 ± 0.129 μM) against Aβ₄₂ aggregation, compared to the reference compound curcumin (95.14% inhibition, IC₅₀ = 6.385 ± 0.009 μM). Compound 6n disassembled preformed Aβ₄₂ aggregates as effectively as curcumin. Furthermore, 6n displayed metal chelating ability and significantly inhibited Cu²⁺-induced Aβ₄₂ aggregation and disassembled preformed Cu²⁺-induced Aβ₄₂ aggregates. 6n successfully controlled the generation of the reactive oxygen species (ROS) by preventing the copper redox cycle. In addition, 6n did not display cytotoxicity and was able to inhibit toxicity induced by Aβ₄₂ aggregates in SH-SY5Y cells. The preferred binding regions and key interactions of 6n with Aβ₄₂ monomer and Aβ₄₂ protofibril structure was evaluated with molecular docking. Compound 6n binds preferably to the C-terminal region of Aβ₄₂ that play a critical role in Aβ₄₂ aggregation. The results of the present study highlight a novel triazole-based compound, 6n, as a promising MTDL against AD.     

Charged surfactants induce a non‐fibrillar aggregation pathway of amyloid‐beta peptide

The amyloid β‐peptide with a sequence of 42 amino acids is the major constituent of extracellular amyloid deposits in Alzheimer's disease plaques. The control of the peptide self‐assembly is difficult to achieve because the process is fast and is affected by many variables. In this paper, we describe the effect of different charged and non‐charged surfactants on Aβ_(1‐42) fibrillation to define common alternate aggregation pathways. The characterization of the peptide‐surfactant interactions by ultra‐structural analysis, thioflavin T assay and secondary structure analysis, suggested that charged surfactants interact with Aβ_(1‐42) through electrostatic interactions. Charged micelles slow down the aggregation process and stabilize the peptide in the oligomeric state, whereas non‐charged surfactants promote the Aβ_(1‐42) fibril formation. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

β-turn tendency in N-methylated peptides with dehydrophenylalanine residue: DFT study

The tendency to adopt β‐turn conformation by model dipeptides with α,β‐dehydrophenylalanine (ΔPhe) residue in the gas phase and in solution is investigated by theoretical methods. We pay special attention to a dependence of conformational properties on the side‐chain configuration of dehydro residue and the influence of N‐methylation on β‐turn stability. An extensive computational study of the conformational preferences of Z and E isomers of dipeptides Ac‐Gly‐(E/Z)‐ΔPhe‐NHMe ( 1a / 1b ) and Ac‐Gly‐(E/Z)‐ΔPhe‐NMe₂ ( 2a / 2b ) by B3LYP/6‐311++G(d,p) and MP2/6‐311++G(d,p) methods is reported. It is shown that, in agreement with experimental data, Ac‐Gly‐(Z)‐ΔPhe‐NHMe has a great tendency to adopt β‐turn conformation. In the gas phase the type II β‐turn is preferred, whereas in the polar environment, the type I. On the other hand, dehydro residue in Ac‐Gly‐(E)‐ΔPhe‐NHMe has a preference to adopt extended conformations in all environments. N‐methylation of C‐terminal amide group, which prevents the formation of 1←4 intramolecular hydrogen bond, change dramatically the conformational properties of studied dehydropeptides. Especially, the tendency to adopt β‐turn conformations is much weaker for the N‐methylated Z isomer (Ac‐Gly‐(Z)‐ΔPhe‐NMe₂), both in vacuo and in the polar environment. On the contrary, N‐methylated E isomer (Ac‐Gly‐(E)‐ΔPhe‐NMe₂) can easier adopt β‐turn conformation, but the backbone torsion angles (?₁, ψ₁, ?₂, ψ₂) are off the limits for common β‐turn types. © 2012 Wiley Periodicals, Inc. Biopolymers 97:518–528, 2012.     

His‐tag binding by antibody C706 mimics β‐amyloid recognition

Alzheimer's disease is a progressive neurodegenerative disease characterized by extracellular deposits of β‐amyloid (Aβ) plaques. Aggregation of the Aβ₄₂ peptide leading to plaque formation is believed to play a central role in Alzheimer's disease pathogenesis. Anti‐Aβ monoclonal antibodies can reduce amyloid plaques and could possibly be used for immunotherapy. We have developed a monoclonal antibody C706, which recognizes the human Aβ peptide. Here we report the crystal structure of the antibody Fab fragment at 1.7 Å resolution. The structure was determined in two crystal forms, P2₁ and C2. Although the Fab was crystallized in the presence of Aβ₁₆, no peptide was observed in the crystals. The antigen‐binding site is blocked by the hexahistidine tag of another Fab molecule in both crystal forms. The poly‐His peptide in an extended conformation occupies a crevice between the light and heavy chains of the variable domain. Two consecutive histidines (His4–His5) stack against tryptophan residues in the central pocket of the antigen‐binding surface. In addition, they form hydrogen bonds to the acidic residues at the bottom of the pocket. The mode of his‐tag binding by C706 resembles the Aβ recognition by antibodies PFA1 and WO2. All three antibodies recognize the same immunodominant B‐cell epitope of Aβ. By similarity, residues Phe–Arg–His of Aβ would be a major portion of the C706 epitope. Copyright © 2010 John Wiley & Sons, Ltd.     

N‐terminal truncated pyroglutamyl β amyloid peptide Aβpy3‐42 shows a faster aggregation kinetics than the full‐length Aβ1‐42

We tested directly the differences in the aggregation kinetics of three important β amyloid peptides, the full‐length Aβ1‐42, and the two N‐terminal truncated and pyroglutamil modified Aβpy3‐42 and Aβpy11‐42 found in different relative concentrations in the brains in normal aging and in Alzheimer disease. By following the circular dichroism signal and the ThT fluorescence of the solution in phosphate buffer, we found substantially faster aggregation kinetics for Aβpy3‐42. This behavior is due to the particular sequence of this peptide, which is also responsible for the specific oligomeric aggregation states, found by TEM, during the fibrillization process, which are very different from those of Aβ1‐42, more prone to fibril formation. In addition, Aβpy3‐42 is found here to have an inhibitory effect on Aβ1‐42 fibrillogenesis, coherently with its known greater infective power. This is an indication of the important role of this peptide in the aggregation process of β‐peptides in Alzheimer disease. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 861–873, 2009. This article was originally published online as an accepted preprint. The “Published Online“ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. III. Dynamics of long‐range hydrophobic interactions

A 20‐residue peptide, IG(42–61), derived from the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptoccocus was studied using circular dichroism, nuclear magnetic resonance (NMR) spectroscopy at various temperatures and by differential scanning calorimetry (DSC). Unlike other related peptides studied so far, this peptide displays two heat capacity peaks in DSC measurements (at a scanning rate of 1.5 deg/min at a peptide concentration of 0.07 mM), which suggests a three‐state folding/unfolding process. The results from DSC and NMR measurements suggest the formation of a dynamic network of hydrophobic interactions stabilizing the structure, which resembles a β‐hairpin shape over a wide range of temperatures (283–313 K). Our results show that IG (42–61) possesses a well‐organized three‐dimensional structure stabilized by long‐range hydrophobic interactions (Tyr50 ··· Phe57 and Trp48 ··· Val59) at T = 283 K and (Trp48 ··· Val59) at 305 and 313 K. The mechanism of β‐hairpin folding and unfolding, as well as the influence of peptide length on its conformational properties, are also discussed. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Effect of the ΔPhe residue configuration on a didehydropeptides conformation: A combined CD and NMR study

Conformations of two pairs of dehydropeptides with the opposite configuration of the ΔPhe residue, Boc‐Gly‐Δ^ZPhe‐Gly‐Phe‐OMe ( Z‐ OMe ), Boc‐Gly‐Δ^EPhe‐Gly‐Phe‐OMe ( E‐ OMe ), Boc‐Gly‐Δ^ZPhe‐Gly‐Phe‐p‐NA ( Z‐p‐ NA ), and Boc‐Gly‐Δ^EPhe‐Gly‐Phe‐p‐NA ( E‐p‐ NA ) were compared on the basis of CD and NMR studies in MeOH, trifluoroethanol (TFE), MeCN, chloroform, and dimethylsulfoxide (DMSO). The CD results were used as the additional input data for the NMR‐based determination of the detailed solution conformations of the peptides. It was found that E‐ OMe is unordered and Z‐ OMe , Z‐p‐ NA , and E‐p‐ NA adopt the β‐turn conformation. There are two overlapping β‐turns in each of those peptides: type II and type III′ in Z‐ OMe and Z‐p‐ NA , and two type III in E‐p‐ NA . The ordered structure‐inducing properties of Δ^ZPhe and Δ^EPhe in the peptides studied depend on the C‐terminal blocking group. In methyl esters, the Δ^ZPhe residue is a strong inducer of ordered conformations whereas the Δ^EPhe one has no such properties. In p‐nitroanilides, both isomers of ΔPhe cause the peptides to adopt ordered structures to a similar extent. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1055–1064, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

C-Terminal Turn Stability Determines Assembly Differences between Aβ40 and Aβ42

Oligomerization of the amyloid β-protein (Aβ) is a seminal event in Alzheimer's disease. Aβ42, which is only two amino acids longer than Aβ40, is particularly pathogenic. Why this is so has not been elucidated fully. We report here results of computational and experimental studies revealing a C-terminal turn at Val36–Gly37 in Aβ42 that is not present in Aβ40. The dihedral angles of residues 36 and 37 in an Ile31–Ala42 peptide were consistent with β-turns, and a β-hairpin-like structure was indeed observed that was stabilized by hydrogen bonds and by hydrophobic interactions between residues 31–35 and residues 38–42. In contrast, Aβ(31–40) mainly existed as a statistical coil. To study the system experimentally, we chemically synthesized Aβ peptides containing amino acid substitutions designed to stabilize or destabilize the hairpin. The triple substitution Gly33Val–Val36Pro–Gly38Val (“VPV”) facilitated Aβ42 hexamer and nonamer formation, while inhibiting formation of classical amyloid-type fibrils. These assemblies were as toxic as were assemblies from wild-type Aβ42. When substituted into Aβ40, the VPV substitution caused the peptide to oligomerize similarly to Aβ42. The modified Aβ40 was significantly more toxic than Aβ40. The double substitution d-Pro36–l-Pro37 abolished hexamer and dodecamer formation by Aβ42 and produced an oligomer size distribution similar to that of Aβ40. Our data suggest that the Val36–Gly37 turn could be the sine qua non of Aβ42. If true, this structure would be an exceptionally important therapeutic target.     

Side‐chain hydrophobicity and the stability of Aβ16–22 aggregates

Recent mutagenesis studies using the hydrophobic segment of Aβ suggest that aromatic π‐stacking interactions may not be critical for fibril formation. We have tested this conjecture by probing the effect of Leu, Ile, and Ala mutation of the aromatic Phe residues at positions 19 and 20, on the double‐layer hexametric chains of Aβ fragment Aβ_16–22 using explicit solvent all‐atom molecular dynamics. As these simulations rely on the accuracy of the utilized force fields, we first evaluated the dynamic and stability dependence on various force fields of small amyloid aggregates. These initial investigations led us to choose AMBER99SB‐ILDN as force field in multiple long molecular dynamics simulations of 100 ns that probe the stability of the wild‐type and mutants oligomers. Single‐point and double‐point mutants confirm that size and hydrophobicity are key for the aggregation and stability of the hydrophobic core region (Aβ_16–22). This suggests as a venue for designing Aβ aggregation inhibitors the substitution of residues (especially, Phe 19 and 20) in the hydrophobic region (Aβ_16–22) with natural and non‐natural amino acids of similar size and hydrophobicity.