Probing the Influence of Single-Site Mutations in the Central Cross-ß Region of Amyloid ß (1-40) Peptides.

Amyloid ß (Aß) is a peptide known to form amyloid fibrils in the brain of patients suffering from Alzheimer's disease. A complete mechanistic understanding how Aß peptides form neurotoxic assemblies and how they kill neurons has not yet been achieved. Previous analysis of various Aß40 mutants could reveal the significant importance of the hydrophobic contact between the residues Phe19 and Leu34 for cell toxicity. For some mutations at Phe19, toxicity was completely abolished. In the current study, we assessed if perturbations introduced by mutations in the direct proximity of the Phe19/Leu34 contact would have similar relevance for the fibrillation kinetics, structure, dynamics and toxicity of the Aß assemblies. To this end, we rationally modified positions Phe20 or Gly33. A small library of Aß40 peptides with Phe20 mutated to Lys, Tyr or the non-proteinogenic cyclohexylalanine (Cha) or Gly33 mutated to Ala was synthesized. We used electron microscopy, circular dichroism, X-ray diffraction, solid-state NMR spectroscopy, ThT fluorescence and MTT cell toxicity assays to comprehensively investigate the physicochemical properties of the Aß fibrils formed by the modified peptides as well as toxicity to a neuronal cell line. Single mutations of either Phe20 or Gly33 led to relatively drastic alterations in the Aß fibrillation kinetics but left the global, as well as the local structure, of the fibrils largely unchanged. Furthermore, the introduced perturbations caused a severe decrease or loss of cell toxicity compared to wildtype Aß40. We suggest that perturbations at position Phe20 and Gly33 affect the fibrillation pathway of Aß40 and, thereby, influence the especially toxic oligomeric species manifesting so that the region around the Phe19/Leu34 hydrophobic contact provides a promising site for the design of small molecules interfering with the Aß fibrillation pathway.

Floquet theory in magnetic resonance: Formalism and applications.

Floquet theory is an elegant mathematical formalism originally developed to solve time-dependent differential equations. Besides other fields, it has found applications in optical spectroscopy and nuclear magnetic resonance (NMR). This review attempts to give a perspective of the Floquet formalism as applied in NMR and shows how it allows one to solve various problems with a focus on solid-state NMR. We include both matrix- and operator-based approaches. We discuss different problems where the Hamiltonian changes with time in a periodic way. Such situations occur, for example, in solid-state NMR experiments where the time dependence of the Hamiltonian originates either from magic-angle spinning or from the application of amplitude- or phase-modulated radiofrequency fields, or from both. Specific cases include multiple-quantum and multiple-frequency excitation schemes. In all these cases, Floquet analysis allows one to define an effective Hamiltonian and, moreover, to treat cases that cannot be described by the more popularly used and simpler-looking average Hamiltonian theory based on the Magnus expansion. An important example is given by spin dynamics originating from multiple-quantum phenomena (level crossings). We show that the Floquet formalism is a very general approach for solving diverse problems in spectroscopy.

Multiplexing experiments in NMR and multi-nuclear MRI.

Multiplexing NMR experiments by direct detection of multiple free induction decays (FIDs) in a single experiment offers a dramatic increase in the spectral information content and often yields significant improvement in sensitivity per unit time. Experiments with multi-FID detection have been designed with both homonuclear and multinuclear acquisition, and the advent of multiple receivers on commercial spectrometers opens up new possibilities for recording spectra from different nuclear species in parallel. Here we provide an extensive overview of such techniques, designed for applications in liquid- and solid-state NMR as well as in hyperpolarized samples. A brief overview of multinuclear MRI is also provided, to stimulate cross fertilization of ideas between the two areas of research (NMR and MRI). It is shown how such techniques enable the design of experiments that allow structure elucidation of small molecules from a single measurement. Likewise, in biomolecular NMR experiments multi-FID detection allows complete resonance assignment in proteins. Probes with multiple RF microcoils routed to multiple NMR receivers provide an alternative way of increasing the throughput of modern NMR systems, effectively reducing the cost of NMR analysis and increasing the information content at the same time. Solid-state NMR experiments have also benefited immensely from both parallel and sequential multi-FID detection in a variety of multi-dimensional pulse schemes. We are confident that multi-FID detection will become an essential component of future NMR methodologies, effectively increasing the sensitivity and information content of NMR measurements.

Experiments with direct detection of multiple FIDs.

Pulse schemes with direct observation of multiple free induction decays (FIDs) offer a dramatic increase in the spectral information content of NMR experiments and often yield substantial improvement in measurement sensitivity per unit time. Availability of multiple receivers on the state-of-the-art commercial spectrometers allows spectra from different nuclear species to be recorded in parallel routinely. Experiments with multi-FID detection have been designed with both, homonuclear and multinuclear acquisition. We provide a brief overview of such techniques designed for applications in liquid- and solid- state NMR as well as in hyperpolarized samples. Here we show how these techniques have led to design of experiments that allow structure elucidation of small molecules and resonance assignment in proteins from a single measurement. Probes with multiple RF micro-coils routed to multiple NMR receivers provide an alternative way of increasing the throughput of modern NMR systems. Solid-state NMR experiments have also benefited immensely from both parallel and simultaneous FID acquisition in a variety of multi-dimensional pulse schemes. We believe that multi-FID detection will become an essential component of the future NMR methodologies effectively increasing the information content of NMR experiments and reducing the cost of NMR analysis.

Simultaneous homonuclear and heteronuclear spin decoupling in magic-angle spinning solid-state NMR.

We show here an effective way of implementing simultaneously homonuclear and heteronuclear dipolar decoupling in magic-angle spinning (MAS) solid-state NMR. Whilst the homonuclear spin decoupling is applied on the 1H channel, heteronuclear spin decoupling is applied on the 13C channel. The 1H spins are observed in a windowed fashion in this case. The resultant 1H spectrum has higher resolution due to the attenuation of broadening arising from both homonuclear 1H-1H and heteronuclear 1H-13C interactions, with the latter normally leading to additional line broadening in 13C labelled samples. The experiments are performed at MAS frequencies of ca. 60 kHz.

Major Reaction Coordinates Linking Transient Amyloid-ß Oligomers to Fibrils Measured at Atomic Level.

The structural underpinnings for the higher toxicity of the oligomeric intermediates of amyloidogenic peptides, compared to the mature fibrils, remain unknown at present. The transient nature and heterogeneity of the oligomers make it difficult to follow their structure. Here, using vibrational and solid-state nuclear magnetic resonance spectroscopy, and molecular dynamics simulations, we show that freely aggregating Aß40 oligomers in physiological solutions have an intramolecular antiparallel configuration that is distinct from the intermolecular parallel ß-sheet structure observed in mature fibrils. The intramolecular hydrogen-bonding network flips nearly 90°, and the two ß-strands of each monomeric unit move apart, to give rise to the well-known intermolecular in-register parallel ß-sheet structure in the mature fibrils. Solid-state nuclear magnetic resonance distance measurements capture the interstrand separation within monomer units during the transition from the oligomer to the fibril form. We further find that the D23-K28 salt-bridge, a major feature of the Aß40 fibrils and a focal point of mutations linked to early onset Alzheimer's disease, is not detectable in the small oligomers. Molecular dynamics simulations capture the correlation between changes in the D23-K28 distance and the flipping of the monomer secondary structure between antiparallel and parallel ß-sheet architectures. Overall, we propose interstrand separation and salt-bridge formation as key reaction coordinates describing the structural transition of the small Aß40 oligomers to fibrils.

Curcumin Dictates Divergent Fates for the Central Salt Bridges in Amyloid-ß40 and Amyloid-ß42.

There are three specific regions in the Amyloid beta (Aß) peptide sequence where variations cause enhanced toxicity in Alzheimer's disease: the N-terminus, the central salt bridge, and the C-terminus. Here, we investigate if there is a close conformational connection between these three regions, which may suggest a concerted mechanism of toxicity. We measure the effects of Zn2+ and curcumin on Aß40, and compare these with their previously reported effects on Aß42. Aß42 and Aß40 differ only near the C-terminus, where curcumin interacts, while Zn2+ interacts near the N-terminus. Therefore, this comparison should help us differentiate the effect of modulating the C- and the N-termini. We find that curcumin allows fibril-like structures containing the salt bridge to emerge in the mature Aß40 aggregates, but not in Aß42. In contrast, we find no difference in the effects of Zn+2 on Aß40 and Aß42. In the presence of Zn+2, both of these fail to form proper fibrils, and the salt bridge remains disrupted. These results indicate that modulations of the Aß termini can determine the fate of a salt bridge far away in the sequence, and this has significant consequences for Aß toxicity. We also infer that small molecules can alter oligomer-induced toxicity by modulating the aggregation pathway, without substantially changing the final product of aggregation.

A suite of pulse sequences based on multiple sequential acquisitions at one and two radiofrequency channels for solid-state magic-angle spinning NMR studies of proteins.

One of the fundamental challenges in the application of solid-state NMR is its limited sensitivity, yet a majority of experiments do not make efficient use of the limited polarization available. The loss in polarization in a single acquisition experiment is mandated by the need to select out a single coherence pathway. In contrast, sequential acquisition strategies can encode more than one pathway in the same experiment or recover unused polarization to supplement a standard experiment. In this article, we present pulse sequences that implement sequential acquisition strategies on one and two radiofrequency channels with a combination of proton and carbon detection to record multiple experiments under magic-angle spinning. We show that complementary 2D experiments such as [Formula: see text] and [Formula: see text] or DARR and [Formula: see text], and 3D experiments such as [Formula: see text] and [Formula: see text], or [Formula: see text] and [Formula: see text]  can be combined in a single experiment to ensure time savings of at least 40 %. These experiments can be done under fast or slow-moderate magic-angle spinning frequencies aided by windowed [Formula: see text] acquisition and homonulcear decoupling. The pulse sequence suite is further expanded by including pathways that allow the recovery of residual polarization, the so-called 'afterglow' pathways, to encode a number of pulse sequences to aid in assignments and chemical-shift mapping.

A Detailed Analysis of the Morphology of Fibrils of Selectively Mutated Amyloid ß (1-40).

A small library of rationally designed amyloid ß [Aß(1-40)] peptide variants is generated, and the morphology of their fibrils is studied. In these molecules, the structurally important hydrophobic contact between phenylalanine 19 (F19) and leucine 34 (L34) is systematically mutated to introduce defined physical forces to act as specific internal constraints on amyloid formation. This Aß(1-40) peptide library is used to study the fibril morphology of these variants by employing a comprehensive set of biophysical techniques including solution and solid-state NMR spectroscopy, AFM, fluorescence correlation spectroscopy, and XRD. Overall, the findings demonstrate that the introduction of significant local physical perturbations of a crucial early folding contact of Aß(1-40) only results in minor alterations of the fibrillar morphology. The thermodynamically stable structure of mature Aß fibrils proves to be relatively robust against the introduction of significantly altered molecular interaction patterns due to point mutations. This underlines that amyloid fibril formation is a highly generic process in protein misfolding that results in the formation of the thermodynamically most stable cross-ß structure.

Positron annihilation and nuclear magnetic resonance study of the phase behavior of water confined in mesopores at different levels of hydration.

We investigated the molecular origin of the phase behavior of water confined in MCM 41 mesopores at different levels of hydration using positron annihilation spectroscopic and nuclear magnetic resonance techniques. The level of hydration influenced the phase behavior of the nanoconfined water. Two transitions above and below the bulk freezing temperature were observed depending on the level of hydration. At the highest level of hydration, nucleation seemed to predominate over the effect of confinement, leading to the complete freezing of water, whereas disrupted H-bonding dominated at the lowest level of hydration, leading to the disappearance of the transitions. A transition at c. T = 188 K (close to the reported glass transition temperature of interface-affected water) was observed at intermediate hydration level. This study suggests that the H-bonding network within nanoconfined water, which can be tampered by the degree of hydration, is the key factor responsible for the phase behavior of supercooled water. This study on the phase behavior and associated transitions of nanoconfined water has implications for nanofluidics and drug-delivery systems, in addition to understanding the fundamentals of water in confinement.

Proton-detected solid-state NMR spectroscopy of fully protonated proteins at slow to moderate magic-angle spinning frequencies.

(1)H-detection offers a substitute to the sensitivity-starved experiments often used to characterize biomolecular samples using magic-angle spinning solid-state NMR spectroscopy (MAS-ssNMR). To mitigate the effects of the strong (1)H-(1)H dipolar coupled network that would otherwise severely broaden resonances, high MAS frequencies (>40kHz) are often employed. Here, we have explored the alternative of stroboscopic (1)H-detection at moderate MAS frequencies of 5-30kHz using windowed version of supercycled-phase-modulated Lee-Goldburg homonuclear decoupling. We show that improved resolution in the (1)H dimension, comparable to that obtainable at high spinning frequencies of 40-60kHz without homonuclear decoupling, can be obtained in these experiments for fully protonated proteins. Along with detailed analysis of the performance of the method on the standard tri-peptide f-MLF, experiments on micro-crystalline GB1 and amyloid-ß aggregates are used to demonstrate the applicability of these pulse-sequences to challenging biomolecular systems. With only two parameters to optimize, broadbanded performance of the homonuclear decoupling sequence, linear dependence of the chemical-shift scaling factor on resonance offset and a straightforward implementation under experimental conditions currently used for many biomolecular studies (viz. spinning frequencies and radio-frequency amplitudes), we expect these experiments to complement the current (13)C-detection based methods in assignments and characterization through chemical-shift mapping.

Steric Crowding of the Turn Region Alters the Tertiary Fold of Amyloid-ß18-35 and Makes It Soluble.

Aß self-assembles into parallel cross-ß fibrillar aggregates, which is associated with Alzheimer's disease pathology. A central hairpin turn around residues 23-29 is a defining characteristic of Aß in its aggregated state. Major biophysical properties of Aß, including this turn, remain unaltered in the central fragment Aß18-35. Here, we synthesize a single deletion mutant, ΔG25, with the aim of sterically hindering the hairpin turn in Aß18-35. We find that the solubility of the peptide goes up by more than 20-fold. Although some oligomeric structures do form, solution state NMR spectroscopy shows that they have mostly random coil conformations. Fibrils ultimately form at a much higher concentration but have widths approximately twice that of Aß18-35, suggesting an opening of the hairpin bend. Surprisingly, two-dimensional solid state NMR shows that the contact between Phe(19) and Leu(34) residues, observed in full-length Aß and Aß18-35, is still intact in these fibrils. This is possible if the monomers in the fibril are arranged in an antiparallel ß-sheet conformation. Indeed, IR measurements, supported by tyrosine cross-linking experiments, provide a characteristic signature of the antiparallel ß-sheet. We conclude that the self-assembly of Aß is critically dependent on the hairpin turn and on the contact between the Phe(19) and Leu(34) regions, making them potentially sensitive targets for Alzheimer's therapeutics. Our results show the importance of specific conformations in an aggregation process thought to be primarily driven by nonspecific hydrophobic interactions.

Cell-Membrane-Mimicking Lipid-Coated Nanoparticles Confer Raman Enhancement to Membrane Proteins and Reveal Membrane-Attached Amyloid-ß Conformation.

Identifying the structures of membrane bound proteins is critical to understanding their function in healthy and diseased states. We introduce a surface enhanced Raman spectroscopy technique which can determine the conformation of membrane-bound proteins, at low micromolar concentrations, and also in the presence of a substantial membrane-free fraction. Unlike conventional surface enhanced Raman spectroscopy, our approach does not require immobilization of molecules, as it uses spontaneous binding of proteins to lipid bilayer-encapsulated Ag nanoparticles. We apply this technique to probe membrane-attached oligomers of Amyloid-ß40 (Aß40), whose conformation is keenly sought in the context of Alzheimer's disease. Isotope-shifts in the Raman spectra help us obtain secondary structure information at the level of individual residues. Our results show the presence of a ß-turn, flanked by two ß-sheet regions. We use solid-state NMR data to confirm the presence of the ß-sheets in these regions. In the membrane-attached oligomer, we find a strongly contrasting and near-orthogonal orientation of the backbone H-bonds compared to what is found in the mature, less-toxic Aß fibrils. Significantly, this allows a "porin" like ß-barrel structure, providing a structural basis for proposed mechanisms of Aß oligomer toxicity.

Probing the role of the ceramide acyl chain length and sphingosine unsaturation in model skin barrier lipid mixtures by (2)H solid-state NMR spectroscopy.

We investigated equimolar mixtures of ceramides with lignoceric acid and cholesterol as models for the human stratum corneum by differential scanning calorimetry and (2)H solid-state NMR spectroscopy. Our reference system consisted of lignoceroyl sphingosine (Cer[NS24]), which represents one of the ceramides in the human stratum corneum. Furthermore, the effect of ceramide acyl chain truncation to 16 carbons as in Cer[NS16] and the loss of the C4 trans double bond as in dihydroceramide Cer[NDS24] were studied. Fully relaxed (2)H NMR spectra were acquired for each deuterated component of each mixture separately, allowing the quantitative determination of the individual lipid phases. At skin temperature, the reference system containing Cer[NS24] is characterized by large portions of each component of the mixture in a crystalline phase, which largely restricts the permeability of the skin lipid barrier. The loss of the C4 trans double bond in Cer[NDS24] leads to the replacement of more than 25% of the crystalline phase by an isotropic phase of the dihydroceramide that shows the importance of dihydroceramide desaturation in the formation of the skin lipid barrier. The truncated Cer[NS16] is mostly found in the gel phase at skin temperature, which may explain its negative effect on the transepidermal water loss in atopic dermatitis patients. These significant alterations in the phase behavior of all lipids are further reflected at elevated temperatures. The molecular insights of our study may help us to understand the importance of the structural parameters of ceramides in healthy and compromised skin barriers.

Significant structural differences between transient amyloid-ß oligomers and less-toxic fibrils in regions known to harbor familial Alzheimer's mutations.

Small oligomers of the amyloidâ€…ß (Aß) peptide, rather than the monomers or the fibrils, are suspected to initiate Alzheimer's disease (AD). However, their low concentration and transient nature under physiological conditions have made structural investigations difficult. A method for addressing such problems has been developed by combining rapid fluorescence techniques with slower two-dimensional solid-state NMR methods. The smallest Aß40 oligomers that demonstrate a potential sign of toxicity, namely, an enhanced affinity for cell membranes, were thus probed. The two hydrophobic regions (residues 10-21 and 30-40) have already attained the conformation that is observed in the fibrils. However, the turn region (residues 22-29) and the N-terminal tail (residues 1-9) are strikingly different. Notably, ten of eleven known Aß mutants that are linked to familial AD map to these two regions. Our results provide potential structural cues for AD therapeutics and also suggest a general method for determining transient protein structures.

Curcumin alters the salt bridge-containing turn region in amyloid ß(1-42) aggregates.

Amyloid ß (Aß) fibrillar deposits in the brain are a hallmark of Alzheimer disease (AD). Curcumin, a common ingredient of Asian spices, is known to disrupt Aß fibril formation and to reduce AD pathology in mouse models. Understanding the structural changes induced by curcumin can potentially lead to AD pharmaceutical agents with inherent bio-compatibility. Here, we use solid-state NMR spectroscopy to investigate the structural modifications of amyloid ß(1-42) (Aß42) aggregates induced by curcumin. We find that curcumin induces major structural changes in the Asp-23-Lys-28 salt bridge region and near the C terminus. Electron microscopy shows that the Aß42 fibrils are disrupted by curcumin. Surprisingly, some of these alterations are similar to those reported for Zn(2+) ions, another agent known to disrupt the fibrils and alter Aß42 toxicity. Our results suggest the existence of a structurally related family of quasi-fibrillar conformers of Aß42, which is stabilized both by curcumin and by Zn(2+.)

(13)C-(13)c homonuclear recoupling in solid-state nuclear magnetic resonance at a moderately high magic-angle-spinning frequency.

Two-dimensional (13)C-(13)C correlation experiments are widely employed in structure determination of protein assemblies using solid-state nuclear magnetic resonance. Here, we investigate the process of (13)C-(13)C magnetisation transfer at a moderate magic-angle-spinning frequency of 30 kHz using some of the prominent second-order dipolar recoupling schemes. The effect of isotropic chemical-shift difference and spatial distance between two carbons and amplitude of radio frequency on (1)H channel on the magnetisation transfer efficiency of these schemes is discussed in detail.

Zn(++) binding disrupts the Asp(23)-Lys(28) salt bridge without altering the hairpin-shaped cross-ß Structure of Aß(42) amyloid aggregates.

Observations like high Zn(2+) concentrations in senile plaques found in the brains of Alzheimer's patients and evidences emphasizing the role of Zn(2+) in amyloid-ß (Aß)-induced toxicity have triggered wide interest in understanding the nature of Zn(2+)-Aß interaction. In vivo and in vitro studies have shown that aggregation kinetics, toxicity, and morphology of Aß aggregates are perturbed in the presence of Zn(2+). Structural studies have revealed that Zn(2+) has a binding site in the N-terminal region of monomeric Aß, but not much is precisely known about the nature of binding of Zn(2+) with aggregated forms of Aß or its effect on the molecular structure of these aggregates. Here, we explore this aspect of the Zn(2+)-Aß interaction using one- and two-dimensional (13)C and (15)N solid-state NMR. We find that Zn(2+) causes major structural changes in the N-terminal and the loop region connecting the two ß-sheets. It breaks the salt bridge between the side chains of Asp(23) and Lys(28) by driving these residues into nonsalt-bridge-forming conformations. However, the cross-ß structure of Aß(42) aggregates remains unperturbed though the fibrillar morphology changes distinctly. We conclude that the salt bridge is not important for defining the characteristic molecular architecture of Aß(42) but is significant for determining its fibrillar morphology and toxicity.