Solid-State Nuclear Magnetic Resonance of Copper-Amyloid Beta, Amylospheroids, Fast Magic Angle Spinning

Molecular level structural examination of Cu2+ binding to 40-residue Alzheimer’s amyloid β (Aβ(1-40)) peptides and the resultant production of reactive oxygen species (ROS) was studied by SSNMR and other techniques. Aβ(1-40) peptide is one of the primary component of senile plaques in Alzheimer’s disease (AD) and was shown that Cu ions (400 µM) are accumulated in plaque deposits and bind Aβ peptides. The Cu-Aβ complex is believed to trigger the production of ROS causing oxidative stress. Despite oxidative stress being considered one of the probable mechanisms of AD, detailed binding structure of Cu2+-Aβ fibrils and the molecular level mechanism of the production of ROS in presence of cellular reductants are still largely unknown. In our work, we try to understand the molecular level details of Cu2+ binding to Aβ(1-40) fibrils, the mechanism of ROS production and subsequent binding of Cu ions with Aβ(1-40) fibrils. Initial signal quenching studies by 13C SSNMR and molecular dynamics simulations showed Cu2+ binding to Aβ(1-40) fibrils at Nε in His-13, His-14 and carboxyl groups in Val-40 as well as Glu sidechains (Glu-3,Glu-11, and/or Glu-22). In the presence of physiological amounts of biological reductant ascorbate Cu2+-Aβ(1-40) complex generates ~60 μM H2O2. During the production of H2O2, Cu2+-Aβ(1-40) fibrils undergoes cyclic redox reaction (Cu2+ ↔ Cu+ )- Aβ(1-40) without any alteration to the fibrils. SSNMR experiments revealed that Cu+ ions are bound to Nδ in His-13 and His-14 during the ROS production. In the second part, the site-specific molecular level structural features of the synthetic Aβ(1-42) amylo-spheroids (ASPD) which is a structural/morphological analog of native ASPD extracted from AD patients is discussed. ASPD represents a class of highly cytotoxic and high mass (>100 kDa) spherical aggregates which have distinct surface tertiary structure and induce degeneration of mature neurons through a different mechanism from other non-fibrillar Aβ assemblies and considered to play a primary role in AD. In the last part, sensitivity and resolution enhancement in 13C and 1H SSNMR is demonstrated by paramagnetic relaxation assisted condensed data collection method, ultra-fast MAS (≥ 78kHz) and selective deuteration using stereo array isotope labeling (SAIL) in a high magnetic field

Structural Insight into an Alzheimer’s Brain-Derived Spherical Assembly of Amyloid β by Solid-State NMR

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

Nano-Mole Scale Side-Chain Signal Assignment by ¹H-Detected Protein Solid-State NMR by Ultra-Fast Magic-Angle Spinning and Stereo-Array Isotope Labeling

<div><p>We present a general approach in ¹H-detected ¹³C solid-state NMR (SSNMR) for side-chain signal assignments of 10-50 nmol quantities of proteins using a combination of a high magnetic field, ultra-fast magic-angle spinning (MAS) at ~80 kHz, and stereo-array-isotope-labeled (SAIL) proteins [Kainosho M. <i>et al</i>., Nature <b>440</b>, 52–57, 2006]. First, we demonstrate that ¹H indirect detection improves the sensitivity and resolution of ¹³C SSNMR of SAIL proteins for side-chain assignments in the ultra-fast MAS condition. ¹H-detected SSNMR was performed for micro-crystalline ubiquitin (~55 nmol or ~0.5mg) that was SAIL-labeled at seven isoleucine (Ile) residues. Sensitivity was dramatically improved by ¹H-detected 2D ¹H/¹³C SSNMR by factors of 5.4-9.7 and 2.1-5.0, respectively, over ¹³C-detected 2D ¹H/¹³C SSNMR and 1D ¹³C CPMAS, demonstrating that 2D ¹H-detected SSNMR offers not only additional resolution but also sensitivity advantage over 1D ¹³C detection for the first time. High ¹H resolution for the SAIL-labeled side-chain residues offered reasonable resolution even in the 2D data. A ¹H-detected 3D ¹³C/¹³C/¹H experiment on SAIL-ubiquitin provided nearly complete ¹H and ¹³C assignments for seven Ile residues only within ~2.5 h. The results demonstrate the feasibility of side-chain signal assignment in this approach for as little as 10 nmol of a protein sample within ~3 days. The approach is likely applicable to a variety of proteins of biological interest without any requirements of highly efficient protein expression systems.</p></div

Spinning-speed dependence of 1H MAS spectra of fully protonated and SAIL isoleucine samples.

<p>(a, b) Chemical structures of (a) uniformly ¹³C- and ¹⁵N-labeled (UL) Ile and (b) SAIL-Ile. (c, d) Spinning-speed dependence of ¹H MAS SSNMR spectra of (c) UL-Ile and (d) SAIL-Ile. The peak at 4.8 ppm (*) is likely due to HCl salts.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122714#pone.0122714.ref017" target="_blank">17</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122714#pone.0122714.ref018" target="_blank">18</a>] No window functions were applied.</p

Resolution and side-chain assignments from 3D ¹³C/¹³C/¹H SSNMR of SAIL-Ubq.

<p>(a, b) 2D ¹³C/¹³C 2D projection spectra from a ¹H-detected 3D ¹³C/¹³C/¹H SSNMR of SAIL-Ubq at MAS 80 kHz. All the peaks including minor ones in (a) are attributed to intra-residue cross peaks within the Ile residues. (c–e) Representative 2D ¹³C/¹³C slices corresponding to ¹H chemical shifts of (c) 1.57 ppm, (d) 1.73 ppm, and (e) 1.41 ppm. The data show clear separation of signals for (c) Ile-3, (d) Ile-13, and (e) Ile-44 by ¹H shifts. The spectrum was processed with 45°- and 60°-shifted sine-bell window functions in the ¹H and ¹³C dimensions, respectively. (f) ¹³C/¹H assignments for Ile-61 from the 3D data. The pulse sequence is listed in Fig D in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122714#pone.0122714.s001" target="_blank">S1 File</a>.</p