Comparative molecular dynamics studies of wild-type and oxidized forms of full-length Alzheimer amyloid beta-peptides Abeta(1-40) and Abeta(1-42)

In this study, all-atom 50 ns molecular dynamics simulations are performed on the full-length amyloid beta (Abeta) monomers (WT-Abeta(1-40) and WT-Abeta(1-42)) and their oxidized forms (Met35(O)-Abeta(1-40) and Met35(O)-Abeta(1-42)) in aqueous solution. The effects of the oxidation state of Met35 and the presence of dipeptide (Ile41-Ala42) on the secondary structures of the three distinct regions (the central hydrophobic core region 17-21 (LVFFA), the loop 23-28 (DVGSNK), and the second hydrophobic domain 29-35 (GAIIGLM)) of all monomers have been analyzed in detail, and results are compared with the available experimental information. Our simulations indicate that the WT-Abeta(1-40) monomer adopts an overall beta-hairpin-like structure, which is promoted by the turn region (24-27). This turn region is stabilized through salt-bridge formation between the Asp23 and Lys28 residues. In contrast, the overall structure of the oxidized (Met35(O)-Abeta(1-40)) monomer can be divided into three well-defined bend regions separated by coil segments. These structural differences may be critical for the measured decrease in the rate of aggregation of Met35(O)-Abeta(1-40) peptide. In the WT-Abeta(1-42) monomer, in comparison to the WT-Abeta(1-40), the Asp23-Lys28 salt bridge is absent, and consequently, the turn in the middle (24-27) region has a smaller curvature. The observed difference in the aggregation rates of these two peptides may be related to the opening of the turn (24-27) stabilized by the Asp23-Lys28 salt bridge. For WT-Abeta(1-42), in the absence of this salt bridge, the unfolding and aggregation events may be more favorable than for WT-Abeta(1-40).     

Solution NMR studies of the A beta(1-40) and A beta(1-42) peptides establish that the Met35 oxidation state affects the mechanism of amyloid formation

The pathogenesis of Alzheimer's disease is characterized by the aggregation and fibrillation of the 40-residue A beta(1-40) and 42-residue A beta(1-42) peptides into amyloid plaques. The structural changes associated with the conversion of monomeric A beta peptide building blocks into multimeric fibrillar beta-strand aggregates remain unknown. Recently, we established that oxidation of the methionine-35 side chain to the sulfoxide (Met35(red) --> Met35(ox)) significantly impedes the rate of aggregation and fibrillation of the A beta peptide. To explore this effect at greater resolution, we carefully compared the (1)H, (15)N, and (13)C NMR chemical shifts of four A beta peptides that had the Met35 reduced or oxidized (A beta(1-40)Met35(red), A beta(1-40)Met35(ox), A beta(1-42)Met35(red), and A beta(1-42)Met35(ox)). With the use of a special disaggregation protocol, the highly aggregation prone A beta peptides could be studied at higher, millimolar concentrations (as required by NMR) in aqueous solution at neutral pH, remaining largely monomeric at 5 degrees C as determined by sedimentation equilibrium studies. The NOE, amide-NH temperature coefficients, and chemical shift indices of the (1)H alpha, (13)C alpha, and (13)C beta established that the four peptides are largely random, extended chain structures, with the Met35(ox) reducing the propensity for beta-strand structure at two hydrophobic regions (Leu17-Ala21 and Ile31-Val36), and turn- or bendlike structures at Asp7-Glu11 and Phe20-Ser26. Additional NMR studies monitoring changes that occur during aging at 37 degrees C established that, along with a gradual loss of signal/noise, the Met35(ox) significantly hindered upfield chemical shift movements of the 2H NMR signals for the His6, His13, and His14 side chains. Taken together, the present NMR studies demonstrate that the Met35(red) --> Met35(ox) conversion prevents aggregation by reducing both hydrophobic and electrostatic association and that the A beta(1-40)Met35(red), A beta(1-40)Met35(ox), A beta(1-42)Met35(red), and A beta(1-42)Met35(ox) peptides may associate differently, through specific, sharp changes in structure during the initial stages of aggregation.     

Conformational restriction via cyclization in beta-amyloid peptide Abeta(1-28) leads to an inhibitor of Abeta(1-28) amyloidogenesis and cytotoxicity

The aggregation process of beta-amyloid peptide Abeta into amyloid is strongly associated with the pathology of Alzheimer's disease (AD). Aggregation may involve a transition of an alpha helix in Abeta(1-28) into beta sheets and interactions between residues 18-20 of the "Abeta amyloid core." We applied an i, i+4 cyclic conformational constraint to the Abeta amyloid core and devised side chain-to-side chain lactam-bridged cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28). In contrast to Abeta(1-28) and [Lys(17), Asp(21)]Abeta(1-28), cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) was not able to form beta sheets and cytotoxic amyloid aggregates. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) was able to interact with Abeta(1-28) and to inhibit amyloid formation and cytotoxicity. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) also interacted with Abeta(1-40) and interfered with its amyloidogenesis. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) or similarly constrained Abeta sequences may find therapeutic and diagnostic applications in AD.     

Aβ40和hIAPP在溶液和表面的成核与交叉成核聚集行为

阿尔茨海默氏病(AD)和2型糖尿病(T2DM)是常见的由蛋白质错误折叠引起的疾病，作为与此二者相关的致病蛋白，淀粉样β蛋白(Aβ)和人胰岛淀粉样多肽(hIAPP)的交叉聚集行为暗示了AD和T2DM的相关性。然而，Aβ和hIAPP在体内的交叉聚集过程尚不明确。为了更好地模拟体内环境特征，即同时存在不同形式的淀粉样蛋白聚集体，且少量的聚集体附着在血管壁上会成为聚集过程的种子，本文以硫代黄素T荧光测定，原子力显微镜，圆二色光谱，石英晶体微天平以及MTT法作为研究手段，探究了Aβ和hIAPP在溶液和固体表面的成核与交叉成核聚集行为。结果表明，少量的Aβ40和hIAPP种子(单体浓度的1/50)即可显著改变异源聚集的聚集路径，形成具有不同形态且含有更多β-折叠结构的异源聚集体，导致更高的细胞毒性。溶液和固体表面上的结果均证明异源成核聚集效率低于同源聚集，且异源聚集的特征很大程度上取决于种子类型。此外，不同于溶液中所得结果，hIAPP种子在固体表面的交叉成核聚集效率显著高于Aβ40种子，证明了界面性质对交叉聚集过程的影响。这些结论对于理解淀粉样蛋白交叉聚集过程具有重要意义。     

Analysis of Amyloid Beta Aggregation Kinetics Using Sym‐Triazine β‐Sheet Inhibitors

AD (Alzheimer’s disease) is a progressive neurodegenerative disorder characterized by the cerebral accumulation of fibrillar amyloid‐beta (Aβ) aggregates. Here we present the electrochemistry of two novel sym‐triazine derivatives (TAE‐1, TAE‐2) as modulators of Aβ_1–42 aggregation in vitro. Incubation studies conducted at physiological conditions demonstrated strong inhibition of β‐sheet fibril formation. Uniquely, square‐wave voltammetry indicated progressive changes in the surface‐availability of amyloid‐intercalated triazines for oxidation, mediated by competing peptide self‐assembly. Time‐resolved voltammetric analysis showed increasing anodic peak currents (≥3‐fold) and progressive shifts in redox potentials, measured over 24 h. The more potent aggregation modulator (TAE‐2) showed prolonged association during the pre‐nucleation states of Aβ.     

Hydrogen exchange studies on Alzheimer's amyloid-beta peptides by mass spectrometry using matrix-assisted laser desorption/ionization and electrospray ionization

The conformation and aggregation behavior of synthetic Alzheimer's amyloid peptides (Abeta) has been investigated using hydrogen-deuterium exchange measured by electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. Mass spectrometric fragmentation of deuterated Abeta peptides was carried out by collision-induced dissociation, inlet fragmentation, and post-source decay. In contrast to the C-terminally truncated peptides Abeta(1-40) and Abeta(1-36) showing full hydrogen-deuterium exchange, Abeta(1-42) and the pyroglutamyl peptide Pyr(3)-Abeta(3-42) produced more complex signal patterns resulting from the formation of beta-sheet-structured oligomers having 18-20 strongly protected protons. Using mass spectrometric fragmentation the results show that the reduced isotope exchange of Abeta(1-42) can be attributed to the central part of the chain comprising residues 8-23. This confirms involvement of the hydrophobic binding domain LVFFA in the course of Abeta aggregation and demonstrates that hydrogen-deuterium exchange in combination with mass spectrometry is well suited for structural analysis of monomeric and reversibly associated amyloid peptides using picomole quantities of material.     

The dynamic nature of amyloid beta (1-40) aggregation

In this paper, we characterize the dynamic nature of the full amyloid beta (1-40) (Aβ (1-40)) aggregates. We labeled the peptide with either 5-carboxytetramethylrhodamine (TAMRA) or with fluorescein-isothiocyanate (FITC). The labeled peptides were mixed after separate fibrillization, and the dynamic changes in the structure of the fibrils were imaged using confocal microscopy. Fluorescence resonance energy transfer (FRET) measurements showed that the Aβ (1-40) peptides detach from and reattach to the fibrils in a biologically relevant timescale (days). With time, the two peptides mix at the molecular level. This process is concentration dependent and occurs primarily in the external parts of the aggregates with a half time between 4 and 7 days. This study shows that the combination of confocal microscopy and FRET analysis is a facile method for studying dynamic processes in supra-molecular aggregates.     

Liquid chromatography/tandem mass spectrometry characterization of oxidized amyloid beta peptides as potential biomarkers of Alzheimer's disease

Alzheimer's disease is characterized by the deposition of senile plaques that consist primarily of amyloid beta peptides. There is substantial evidence that amyloid beta is oxidized in vivo, which has led to the suggestion that oxidative stress is an important mediator of Alzheimer's disease. Metal-catalyzed oxidation can mimic in vivo oxidation of amyloid beta because the metal ion binds to the amino acid residues at the site of oxidation, which then deliver reactive oxygen species to that site. Based on electrospray mass spectrometry, it has been suggested that metal-catalyzed oxidation occurs on histidines-13 and -14. Unfortunately, the amyloid beta peptides provide complex spectra, so it is difficult to definitively characterize the sites of oxidation. Trypsin digestion of both native and oxidized amyloid beta1-16 and amyloid beta1-40 resulted in the formation of tryptic peptides corresponding to amyloid beta6-16, which could be separated by liquid chromatography (LC). Sites of oxidation were then unequivocally characterized as histidine-13 and histidine-14 by LC/tandem mass spectrometric (MS/MS) analysis of the tryptic peptides. The ability to analyze the specific amyloid beta6-16 tryptic fragments derived from full-length amyloid beta peptides will make it possible to determine whether oxidation in vivo occurs at specific histidine residues and/or at other amino acid residues such as methionine-35. Using methodology based on LC/MS/MS it will also be possible to analyze the relative amounts of oxidized peptides and native peptide in cerebrospinal fluid from patients with Alzheimer's disease as biomarkers of oxidative stress.     

Charge regulation phenomenon predicted from the modeling of polypeptide electrophoretic mobilities as a relevant mechanism of amyloid‐beta peptide oligomerization

Electrophoretic mobilities of amyloid‐beta (1‐40) and (1‐42) peptides and their aggregates are modeled to study the amyloidogenic pathway associated with Alzheimer´s Disease. The near molecule pH generated by the intraparticle charge regulation phenomenon during the oligomerization of amyloid‐beta (1‐40) and (1‐42) peptides is evaluated and discussed as a relevant mechanism supporting the “amyloid cascade hypothesis” proposed in the literature. A theoretical framework associated with the oligomerization of amyloid‐beta peptides including simple scaling laws and the consideration of electrokinetic and hydrodynamic global properties of oligomers is presented. The central finding is the explanation of the near molecule pH change toward the pI when the oligomerization number increases. These results allow one to rationalize consecutive physical stages that validate the amyloid cascade hypothesis. Concluding remarks involving mainly the effects of pair and intraparticle charge regulation phenomena on the amyloidogenic pathway with some suggestions for future research are provided.     

Why Is the C-terminus of Abeta(1-42) more unfolded than that of Abeta(1-40)? Clues from hydrophobic interaction

Abeta(1-40) and Abeta(1-42) are the main forms of amyloid beta (Abeta) peptides in the brain of Alzheimer's patients; however, the latter possesses much stronger aggregation and deposition propensity than the former, which is partially attributed to the more unfolded C-terminus of Abeta(1-42) than that of Abeta(1-40). To explore the physical basis underlying the different dynamic behaviors of both Abeta peptides, parallel molecular dynamics (MD) simulations on Abeta(1-40) and Abeta(1-42) were performed to investigate their thermal unfolding processes. It is revealed that the addition of residues 41 and 42 in Abeta(1-42) disrupts the C-terminal hydrophobic core, which triggers the unraveling of the C-terminal helix of Abeta(1-42). This conclusion is supported by the MD simulation on the I41A mutant of Abeta(1-42), in which the C-terminal helix possesses relatively higher conformational stability than that of wild type Abeta(1-42) owing to the change in hydrophobic interaction patterns.     

Dynamics of Zn(II) binding as a key feature in the formation of amyloid fibrils by Aβ11-28

Supramolecular assembly of peptides and proteins into amyloid fibrils is of multifold interest, going from materials science to physiopathology. The binding of metal ions to amyloidogenic peptides is associated with several amyloid diseases, and amyloids with incorporated metal ions are of interest in nanotechnology. Understanding the mechanisms of amyloid formation and the role of metal ions can improve strategies toward the prevention of this process and enable potential applications in nanotechnology. Here, studies on Zn(II) binding to the amyloidogenic peptide Aβ11-28 are reported. Zn(II) modulates the Aβ11-28 aggregation, in terms of kinetics and fibril structures. Structural studies suggest that Aβ11-28 binds Zn(II) by amino acid residues Glu11 and His14 and that Zn(II) is rapidly exchanged between peptides. Structural and aggregation data indicate that Zn(II) binding induces the formation of the dimeric Zn(II)(1)(Aβ11-28)(2) species, which is the building block of fibrillar aggregates and explains why Zn(II) binding accelerates Aβ11-28 aggregation. Moreover, transient Zn(II) binding, even briefly, was enough to promote fibril formation, but the final structure resembled that of apo-Aβ11-28 amyloids. Also, seeding experiments, i.e., the addition of fibrillar Zn(II)(1)(Aβ11-28)(2) to the apo-Aβ11-28 peptide, induced aggregation but not propagation of the Zn(II)(1)(Aβ11-28)(2)-type fibrils. This can be explained by the dynamic Zn(II) binding between soluble and aggregated Aβ11-28. As a consequence, dynamic Zn(II) binding has a strong impact on the aggregation behavior of the Aβ11-28 peptide and might be a relevant and so far little regarded parameter in other systems of metal ions and amyloidogenic peptides.     

Exploring the inter-molecular interactions in amyloid-β protofibril with molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area free energy calculations

Aggregation of amyloid-β (Aβ) peptides correlates with the pathology of Alzheimer's disease. However, the inter-molecular interactions between Aβ protofibril remain elusive. Herein, molecular mechanics Poisson-Boltzmann surface area analysis based on all-atom molecular dynamics simulations was performed to study the inter-molecular interactions in Aβ(17-42) protofibril. It is found that the nonpolar interactions are the important forces to stabilize the Aβ(17-42) protofibril, while electrostatic interactions play a minor role. Through free energy decomposition, 18 residues of the Aβ(17-42) are identified to provide interaction energy lower than -2.5 kcal/mol. The nonpolar interactions are mainly provided by the main chain of the peptide and the side chains of nine hydrophobic residues (Leu17, Phe19, Phe20, Leu32, Leu34, Met35, Val36, Val40, and Ile41). However, the electrostatic interactions are mainly supplied by the main chains of six hydrophobic residues (Phe19, Phe20, Val24, Met35, Val36, and Val40) and the side chains of the charged residues (Glu22, Asp23, and Lys28). In the electrostatic interactions, the overwhelming majority of hydrogen bonds involve the main chains of Aβ as well as the guanidinium group of the charged side chain of Lys28. The work has thus elucidated the molecular mechanism of the inter-molecular interactions between Aβ monomers in Aβ(17-42) protofibril, and the findings are considered critical for exploring effective agents for the inhibition of Aβ aggregation.     

Estimation of electrokinetic and hydrodynamic global properties of relevant amyloid‐beta peptides through the modeling of their effective electrophoretic mobilities and analysis of their propensities to aggregation

Neuronal activity loss may be due to toxicity caused by amyloid‐beta peptides forming soluble oligomers. Here amyloid‐beta peptides (1–42, 1–40, 1–39, 1–38, and 1–37) are characterized through the modeling of their experimental effective electrophoretic mobilities determined by a capillary zone electrophoresis method as reported in the literature. The resulting electrokinetic and hydrodynamic global properties are used to evaluate amyloid‐beta peptide propensities to aggregation through pair particles interaction potentials and Brownian aggregation kinetic theories. Two background electrolytes are considered at 25°C, one for pH 9 and ionic strength I = 40 mM (aggregation is inhibited through NH₄OH) the other for pH 10 and I = 100 mM (without NH₄OH). Physical explanations of peptide oligomerization mechanisms are provided. The effect of hydration, electrostatic, and dispersion forces in the amyloidogenic process of amyloid‐beta peptides (1–40 and 1–42) are quantitatively presented. The interplay among effective charge number, hydration, and conformation of chains is described. It is shown that amyloid‐beta peptides (1–40 and 1–42) at pH 10, I = 100 mM and 25°C, may form soluble oligomers, mainly of order 2 and 4, after an incubation of 48 h, which at higher times evolve and end up in complex structures (protofibrils and fibrils) found in plaques associated with Alzheimer's disease.     

Effect of freezing on amyloid peptide aggregation and self-diffusion in an aqueous solution

Pulsed-field gradient ¹H NMR is employed to investigate the self-diffusion of amyloid Aβ-peptide in an aqueous buffer solution (pH 7.44) with a protein concentration of 50 μmol at 20°C. The self-diffusion coefficient of the peptide in a freshly prepared solution corresponds to its monomeric form. The storage of the solution at 24°C causes part of the peptide molecules to form amyloid aggregates as soon as over 48 h. However, the ¹H NMR echo signal typical of aggregated molecules is not observed because of their dense packing in the aggregates and a large mass of the latter. A freezing-fusion of the solution after the aggregation does not cause changes in the self-diffusion coefficients of the peptide. After a peptide solution free of amyloid aggregates is subjected to a freezing-fusion cycle, part of the peptide molecules also remains in the monomeric form in the solution, while another part forms amyloid aggregates, with a portion of the aggregated peptide molecules retaining a high rotational mobility with virtually absolute absence of a translational mobility. The results obtained are interpreted in terms of the formation of “porous aggregates” of amyloid fibrils, with “pores” having sizes comparable with those of peptide molecules, though, being larger than water molecules. Peptide molecules, which do not form fibrils, are captured in the pores. Temperature regime is shown to be of importance for the aggregation of amyloid peptides. In particular, freezing, which is traditionally considered to be a method for the prevention from or temporary interruption of aggregation, may itself lead to the formation of amorphous amyloid aggregates, which remain preserved in solutions after their unfreezing.     

All-atom molecular dynamics studies of the full-length β-amyloid peptides

β-Amyloid peptides are believed to play an essential role in Alzheimer’s disease (AD), due to their sedimentation in the form of β-amyloid aggregates in the brain of AD-patients, and the in vitro neurotoxicity of oligomeric aggregates. The monomeric peptides come in different lengths of 39–43 residues, of which the 42 alloform seems to be most strongly associated with AD-symptoms. Structural information on these peptides to date comes from NMR studies in acidic solutions, organic solvents, or on shorter fragments of the peptide. In addition X-ray and solid-state NMR investigations of amyloid fibrils yield insight into the structure of the final aggregate and therefore define the endpoint of any conformational change of an Aβ-monomer along the aggregation process. The conformational changes necessary to connect the experimentally known conformations are not yet understood and this process is an active field of research.     

Use of 18O labels to monitor deamidation during protein and peptide sample processing

Nonenzymatic deamidation of asparagine residues in proteins generates aspartyl (Asp) and isoaspartyl (isoAsp) residues via a succinimide intermediate in a neutral or basic environment. Electron capture dissociation (ECD) can differentiate and quantify the relative abundance of these isomeric products in the deamidated proteins. This method requires the proteins to be digested, usually by trypsin, into peptides that are amenable to ECD. ECD of these peptides can produce diagnostic ions for each isomer; the c. + 58 and z - 57 fragment ions for the isoAsp residue and the fragment ion ((M + nH)((n-1)+.) - 60) corresponding to the side-chain loss from the Asp residue. However, deamidation can also occur as an artifact during sample preparation, particularly when using typical tryptic digestion protocols. With 18O labeling, it is possible to differentiate deamidation occurring during trypsin digestion which causes a +3 Da (18O1 + 1D) mass shift from the pre-existing deamidation, which leads to a +1-Da mass shift. This paper demonstrates the use of (18)O labeling to monitor three rapidly deamidating peptides released from proteins (calmodulin, ribonuclease A, and lysozyme) during the time course of trypsin digestion processes, and shows that the fast (approximately 4 h) trypsin digestion process generates no additional detectable peptide deamidations.     

Real‐time Amyloid Aggregation Monitoring with a Photonic Crystal‐based Approach

We propose the application of a new label‐free optical technique based on photonic nanostructures to real‐time monitor the amyloid‐beta 1‐42 (Aβ(1‐42)) fibrillization, including the early stages of the aggregation process, which are related to the onset of the Alzheimer’s Disease (AD). The aggregation of Aβ peptides into amyloid fibrils has commonly been associated with neuronal death, which culminates in the clinical features of the incurable degenerative AD. Recent studies revealed that cell toxicity is determined by the formation of soluble oligomeric forms of Aβ peptides in the early stages of aggregation. At this phase, classical amyloid detection techniques lack in sensitivity. Upon a chemical passivation of the sensing surface by means of polyethylene glycol, the proposed approach allows an accurate, real‐time monitoring of the refractive index variation of the solution, wherein Aβ(1‐42) peptides are aggregating. This measurement is directly related to the aggregation state of the peptide throughout oligomerization and subsequent fibrillization. Our findings open new perspectives in the understanding of the dynamics of amyloid formation, and validate this approach as a new and powerful method to screen aggregation at early stages.     

Molecular-level examination of Cu2+ binding structure for amyloid fibrils of 40-residue Alzheimer's β by solid-state NMR spectroscopy

Cu(2+) binding to Alzheimer's β (Aβ) peptides in amyloid fibrils has attracted broad attention, as it was shown that Cu ion concentration elevates in Alzheimer's senile plaque and such association of Aβ with Cu(2+) triggers the production of neurotoxic reactive oxygen species (ROS) such as H(2)O(2). However, detailed binding sites and binding structures of Cu(2+) to Aβ are still largely unknown for Aβ fibrils or other aggregates of Aβ. In this work, we examined molecular details of Cu(2+) binding to amyloid fibrils by detecting paramagnetic signal quenching in 1D and 2D high-resolution (13)C solid-state NMR (SSNMR) for full-length 40-residue Aβ(1-40). Selective quenching observed in (13)C SSNMR of Cu(2+)-bound Aβ(1-40) suggested that primary Cu(2+) binding sites in Aβ(1-40) fibrils include N(ε) in His-13 and His-14 and carboxyl groups in Val-40 as well as in Glu sidechains (Glu-3, Glu-11, and/or Glu-22). (13)C chemical shift analysis demonstrated no major structural changes upon Cu(2+) binding in the hydrophobic core regions (residues 18-25 and 30-36). Although the ROS production via oxidization of Met-35 in the presence of Cu(2+) has been long suspected, our SSNMR analysis of (13)C(ε)H(3)-S- in M35 showed little changes after Cu(2+) binding, excluding the possibility of Met-35 oxidization by Cu(2+) alone. Preliminary molecular dynamics (MD) simulations on Cu(2+)-Aβ complex in amyloid fibrils confirmed binding sites suggested by the SSNMR results and the stabilities of such bindings. The MD simulations also indicate the coexistence of a variety of Cu(2+)-binding modes unique in Aβ fibril, which are realized by both intra- and intermolecular contacts and highly concentrated coordination sites due to the in-register parallel β-sheet arrangements.     

An application of 1H nuclear magnetic resonance to the determination of hydrophobic interactions in peptides

Intermolecular hydrophobic interactions between the indole or phenyl moieties of the peptides containing L -tryptophan (L -Trp) or L -phenylalanine (L -Phe) residues and the apolar isopropyl groups of the peptides containing L -leucine (L -Leu) or L -valine (L -Val) in aqueous solutions have been detected by ¹H NMR by monitoring the observed changes in the proton magnetic resonance parameters of the methyl resonances of the peptides containing L -Leu or L -Val residues. The ¹H NMR data indicate that intermolecular hydrophobic interactions are responsible for the observed changes in the proton magnetic resonance parameters of the methyl resonances. For example, when a solution of glycylglycylleucine (Gly-Gly-L -Leu) in deuterium oxide was mixed with glycylglycyltryptophan (Gly-Gly-L -Trp), the methyl protons of Gly-Gly-L -Leu exhibited large diamagnetic ring current shifts. However, when glycylglycylglycine (Gly-Gly-Gly) was substituted for Gly-Gly-L -Trp in the NMR experiment, the methyl resonances did not show any upfield or downfield shift, thereby demonstrating that the observed upfield shifts are not due to bulk susceptibility differences between solutions. The C-terminus peptides containing L -Leu or L -Val residues bind to the aromatic L -Trp or L -Phe peptides better than the N-terminus L -Leu or L -Val peptides. The C-terminus Gly-Gly-L -Leu binds better than the C-terminus glycylglycylvaline (Gly-Gly-L -Val). The strength and specificity of hydrophobic interactions in several small peptides are discussed.     

Intramolecular Charge Interactions as a Tool to Control the Coiled‐Coil‐to‐Amyloid Transformation

Under the influence of a changed environment, amyloid‐forming proteins partially unfold and assemble into insoluble β‐sheet rich fibrils. Molecular‐level characterization of these assembly processes has been proven to be very challenging, and for this reason several simplified model systems have been developed over recent years. Herein, we present a series of three de novo designed model peptides that adopt different conformations and aggregate morphologies depending on concentration, pH value, and ionic strength. The design strictly follows the characteristic heptad repeat of the α‐helical coiled‐coil structural motif. In all peptides, three valine residues, known to prefer the β‐sheet conformation, have been incorporated at the solvent‐exposed b, c, and f positions to make the system prone to amyloid formation. Additionally, pH‐controllable intramolecular electrostatic repulsions between equally charged lysine (peptide A) or glutamate (peptide B) residues were introduced along one side of the helical cylinder. The conformational behavior was monitored by circular dichroism spectroscopic analysis and thioflavin T fluorescence, and the resulting aggregates were further characterized by transmission electron microscopy. Whereas uninterrupted α‐helical aggregates are found at neutral pH, Coulomb repulsions between lysine residues in peptide A destabilize the helical conformation at acidic pH values and trigger an assembly into amyloid‐like fibrils. Peptide B features a glutamate‐based switch functionality and exhibits opposite pH‐dependent folding behavior. In this case, α‐helical aggregates are found under acidic conditions, whereas amyloids are formed at neutral pH. To further validate the pH switch concept, peptide C was designed by including serine residues, thus resulting in an equal distribution of charged residues. Surprisingly, amyloid formation is observed at all pH values investigated for peptide C. The results of further investigations into the effect of different salts, however, strongly support the crucial role of intramolecular charge repulsions in the model system presented herein.