Accumulating evidence suggests that various neurodegenerative diseases, including Alzheimer’s disease (AD), are linked to cytotoxic diffusible aggregates of amyloid proteins, which are metastable intermediate species in protein misfolding. This study presents the first site-specific structural study on an intermediate called amylospheroid (ASPD), an AD-derived neurotoxin composed of oligomeric amyloid-β (Aβ). Electron microscopy and immunological analyses using ASPD-specific “conformational” antibodies established synthetic ASPD for the 42-residue Aβ(1–42) as an excellent structural/morphological analogue of native ASPD extracted from AD patients, the level of which correlates with the severity of AD. 13C solid-state NMR analyses of approximately 20 residues and interstrand distances demonstrated that the synthetic ASPD is made of a homogeneous single conformer containing parallel β-sheets. These results provide profound insight into the native ASPD, indicating that Aβ is likely to self-assemble into the toxic intermediate with β-sheet structures in AD brains. This approach can be applied to various intermediates relevant to amyloid diseases.

Memory devices are a key technology of our era and one of the constant challenges is the reduction of their power consumption. Herein, we demonstrate that graphene oxide with very few defects, that is, about 1 nm thin oxo-functionalized graphene derivative, can be used in memory devices operating at 3 V. A memory device stores charges in the material of the active channel. Thereby, writing and erasing information can be performed at low voltage, facilitating low power consumption. To enable operation at low voltage, a novel synthetic approach is necessary. We find that the selective non-covalent electrostatic functionalization of mainly organosulfate ions is possible with dodecylammonium. This functionalization allows the non-covalent coating of flakes with a polystyrene-derivative as nm-thin dielectric medium. The resulting polymer-wrapped composite has a height of about 5 nm. We find that the thin coating of a few nm is mandatory to make the memory device work at low voltage. Furthermore, a self-assembled monolayer of an imidazolium derivative further enhances the function of the memory device. The prepared composite materials are characterized by state-of-the-art analysis including solid state nuclear magnetic resonance spectroscopy and thermogravimetric analysis coupled with gas chromatography, mass spectroscopy or infrared spectroscopy. Reference experiments prove the importance of the controlled synthesis to enable the function of the memory device.

We present a 3D 1H-detected solid-state NMR (SSNMR) approach for main-chain signal assignments of 10–100 nmol of fully protonated proteins using ultra-fast magic-angle spinning (MAS) at ∼80 kHz by a novel spectral-editing method, which permits drastic spectral simplification. The approach offers ∼110 fold time saving over a traditional 3D 13C-detected SSNMR approach.

•Novel low-power 1H decoupling for CP-MAS using ultra-fast magic angle spinning.•First demonstration of efficient composite-pulse 1H decoupling for rigid solids.•Feasibility of 2D solid-state NMR for 1 nmol of a protein microcrystal sample.This article describes recent trends of high-field solid-state NMR (SSNMR) experiments for small organic molecules and biomolecules using 13C and 15N CPMAS under ultra-fast MAS at a spinning speed (νR) of 80–100 kHz. First, we illustrate major differences between a modern low-power RF scheme using UFMAS in an ultra-high field and a traditional CPMAS scheme using a moderate sample spinning in a lower field. Features and sensitivity advantage of a low-power RF scheme using UFMAS and a small sample coil are summarized for CPMAS-based experiments. Our 1D 13C CPMAS experiments for uniformly 13C- and 15N-labeled alanine demonstrated that the sensitivity per given sample amount obtained at νR of 100 kHz and a 1H NMR frequency (νH) of 750.1 MHz is ~10 fold higher than that of a traditional CPMAS experiment obtained at νR of 20 kHz and νH of 400.2 MHz. A comparison of different 1H-decoupling schemes in CPMAS at νR of 100 kHz for the same sample demonstrated that low-power WALTZ-16 decoupling unexpectedly displayed superior performance over traditional low-power schemes designed for SSNMR such as TPPM and XiX in a range of decoupling field strengths of 5–20 kHz. Excellent 1H decoupling performance of WALTZ-16 was confirmed on a protein microcrystal sample of GB1 at νR of 80 kHz. We also discuss the feasibility of a SSNMR microanalysis of a GB1 protein sample in a scale of 1 nmol to 80 nmol by 1H-detected 2D 15N/1H SSNMR by a synergetic use of a high field, a low-power RF scheme, a paramagnetic-assisted condensed data collection (PACC), and UFMAS.

Recent research in fast magic angle spinning (MAS) methods has drasticallyimproved the resolution and sensitivity of NMR spectroscopy of biomolecules and materials in solids. In this Account, we summarize recent and ongoing developments in this area by presenting 13C and 1H solid-state NMR (SSNMR) studies on paramagnetic systems and biomolecules under fast MAS from our laboratories.First, we describe how very fast MAS (VFMAS) at the spinning speed of at least20 kHz allows us to overcome major difficulties in 1H and 13C high-resolution SSNMR of paramagnetic systems. As a result, we can enhance both sensitivity and resolution by up to a few orders of magnitude. Using fast recycling (∼ms/scan) with short 1H T1 values, we can perform 1H SSNMR microanalysis of paramagnetic systems on the microgram scale with greatly improved sensitivity over that observed for diamagnetic systems. Second, we discuss how VFMAS at a spinning speed greater than ∼40 kHz can enhance the sensitivity and resolution of 13C biomolecular SSNMR measurements. Low-power 1H decoupling schemes under VFMAS offer excellent spectral resolution for 13C SSNMR by nominal 1H RF irradiation at ∼10 kHz. By combining the VFMAS approach with enhanced 1H T1 relaxation by paramagnetic doping, we can achieve extremely fast recycling in modern biomolecular SSNMR experiments. Experiments with 13C-labeled ubiquitin doped with 10 mM Cu-EDTA demonstrate how effectively this new approach, called paramagnetic assisted condensed data collection (PACC), enhances the sensitivity.Lastly, we examine 13C SSNMR measurements for biomolecules under faster MAS at a higher field. Our preliminary 13C SSNMR data of Aβ amyloid fibrils and GB1 microcrystals acquired at 1H NMR frequencies of 750–800 MHz suggest that the combined use of the PACC approach and ultrahigh fields could allow for routine multidimensional SSNMR analyses of proteins at the 50–200 nmol level. Also, we briefly discuss the prospects for studying bimolecules using 13C SSNMR under ultrafast MAS at the spinning speed of ∼100 kHz.

Solid-phase peptide synthesis (SPPS) is a widely used technique in biology and chemistry. However, the synthesis yield in SPPS often drops drastically for longer amino acid sequences, presumably because of the occurrence of incomplete coupling reactions. The underlying cause for this problem is hypothesized to be a sequence-dependent propensity to form secondary structures through protein aggregation. However, few methods are available to study the site-specific structure of proteins or long peptides that are anchored to the solid support used in SPPS. This study presents a novel solid-state NMR (SSNMR) approach to examine protein structure in the course of SPPS. As a useful benchmark, we describe the site-specific SSNMR structural characterization of the 40-residue Alzheimer’s β-amyloid (Aβ) peptide during SPPS. Our 2D 13C/13C correlation SSNMR data on Aβ(1–40) bound to a resin support demonstrated that Aβ underwent excessive misfolding into a highly ordered β-strand structure across the entire amino acid sequence during SPPS. This approach is likely to be applicable to a wide range of peptides/proteins bound to the solid support that are synthesized through SPPS.

Cu2+ 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 Cu2+ triggers the production of neurotoxic reactive oxygen species (ROS) such as H2O2. However, detailed binding sites and binding structures of Cu2+ to Aβ are still largely unknown for Aβ fibrils or other aggregates of Aβ. In this work, we examined molecular details of Cu2+ binding to amyloid fibrils by detecting paramagnetic signal quenching in 1D and 2D high-resolution 13C solid-state NMR (SSNMR) for full-length 40-residue Aβ(1−40). Selective quenching observed in 13C SSNMR of Cu2+-bound Aβ(1−40) suggested that primary Cu2+ binding sites in Aβ(1−40) fibrils include Nε 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). 13C chemical shift analysis demonstrated no major structural changes upon Cu2+ 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 Cu2+ has been long suspected, our SSNMR analysis of 13CεH3−S− in M35 showed little changes after Cu2+ binding, excluding the possibility of Met-35 oxidization by Cu2+ alone. Preliminary molecular dynamics (MD) simulations on Cu2+−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 Cu2+-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.

Cu(II)(phthalocyanine) (CuPc) is broadly utilized as an archetypal molecular semiconductor and is the most widely used blue printing pigment. CuPc crystallizes in six different forms; the chemical and physical properties are substantially modulated by its molecular packing among these polymorphs. Despite the growing importance of this system, spectroscopic identification of different polymorphs for CuPc has posed difficulties. This study presents the first example of spectroscopic distinction of α- and β-forms of CuPc, the most widely used polymorphs, by solid-state NMR (SSNMR) and Raman spectroscopy. 13C high-resolution SSNMR spectra of α- and β-CuPc using very-fast magic angle spinning (VFMAS) at 20 kHz show that hyperfine shifts sensitively reflect polymorphs of CuPc. The experimental results were confirmed by ab initio chemical shift calculations. 13C and 1H SSNMR relaxation times of α- and β-CuPc under VFMAS also showed marked differences, presumably because of the difference in electronic spin correlation times in the two forms. Raman spectroscopy also provided another reliable method of differentiation between the two polymorphs.

We discuss a simple approach to enhance sensitivity for 13C high-resolution solid-state NMR for proteins in microcrystals by reducing 1H T1 relaxation times with paramagnetic relaxation reagents. It was shown that 1H T1 values can be reduced from 0.4–0.8 s to 60–70 ms for ubiquitin and lysozyme in D2O in the presence of 10 mM Cu(II)Na2EDTA without substantial degradation of the resolution in 13C CPMAS spectra. Faster signal accumulation using the shorter 1H T1 attained by paramagnetic doping provided sensitivity enhancements of 1.4–2.9 for these proteins, reducing the experimental time for a given signal-to-noise ratio by a factor of 2.0–8.4. This approach presented here is likely to be applicable to various other proteins in order to enhance sensitivity in 13C high-resolution solid-state NMR spectroscopy.

Amyloid fibrils of Alzheimer’s β-amyloid peptide (Aβ) are a primary component of amyloid plaques, a hallmark of Alzheimer’s disease (AD). Enormous attention has been given to the structural features and functions of Aβ in amyloid fibrils and other type of aggregates in associated with development of AD. This report describes an efficient protocol to express and purify high-quality 40-residue Aβ(1–40), the most abundant Aβ in brains, for structural studies by NMR spectroscopy. Over-expression of Aβ(1–40) with glutathione S-transferase (GST) tag connected by a Factor Xa recognition site (IEGR▾) in Escherichia coli resulted in the formation of insoluble inclusion bodies even with the soluble GST tag. This problem was resolved by efficient recovery of the GST-Aβ fusion protein from the inclusion bodies using 0.5% (w/v) sodium lauroyl sarcosinate as solubilizing agent and subsequent purification by affinity chromatography using a glutathione agarose column. The removal of the GST tag by Factor Xa enzymatic cleavage and purification by HPLC yielded as much as ∼7 mg and ∼1.5 mg of unlabeled Aβ(1–40) and uniformly 15N- and/or 13C-protein Aβ(1–40) from 1 L of the cell culture, respectively. Mass spectroscopy of unlabeled and labeled Aβ and 1H/15N HSQC solution NMR spectrum of the obtained 15N-labeled Aβ in the monomeric form confirmed the expression of native Aβ(1–40). It was also confirmed by electron micrography and solid-state NMR analysis that the purified Aβ(1–40) self-assembles into β-sheet rich amyloid fibrils. To the best of our knowledge, our protocol offers the highest yields among published protocols for production of recombinant Aβ(1–40) samples that are amendable for an NMR-based structural analysis. The protocol may be applied to efficient preparation of other amyloid-forming proteins and peptides that are 13C- and 15N-labeled for NMR experiments.

Memory devices are a key technology of our era and one of the constant challenges is the reduction of their power consumption. Herein, we demonstrate that graphene oxide with very few defects, that is, about 1 nm thin oxo-functionalized graphene derivative, can be used in memory devices operating at 3 V. A memory device stores charges in the material of the active channel. Thereby, writing and erasing information can be performed at low voltage, facilitating low power consumption. To enable operation at low voltage, a novel synthetic approach is necessary. We find that the selective non-covalent electrostatic functionalization of mainly organosulfate ions is possible with dodecylammonium. This functionalization allows the non-covalent coating of flakes with a polystyrene-derivative as nm-thin dielectric medium. The resulting polymer-wrapped composite has a height of about 5 nm. We find that the thin coating of a few nm is mandatory to make the memory device work at low voltage. Furthermore, a self-assembled monolayer of an imidazolium derivative further enhances the function of the memory device. The prepared composite materials are characterized by state-of-the-art analysis including solid state nuclear magnetic resonance spectroscopy and thermogravimetric analysis coupled with gas chromatography, mass spectroscopy or infrared spectroscopy. Reference experiments prove the importance of the controlled synthesis to enable the function of the memory device.

We present a 3D 1H-detected solid-state NMR (SSNMR) approach for main-chain signal assignments of 10–100 nmol of fully protonated proteins using ultra-fast magic-angle spinning (MAS) at ∼80 kHz by a novel spectral-editing method, which permits drastic spectral simplification. The approach offers ∼110 fold time saving over a traditional 3D 13C-detected SSNMR approach.