Nano-mole scale sequential signal assignment by (1)H-detected protein solid-state NMR.

We present a 3D (1)H-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 (13)C-detected SSNMR approach.

Structural Insight into an Alzheimer's Brain-Derived Spherical Assembly of Amyloid ß 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-ß (Aß). Electron microscopy and immunological analyses using ASPD-specific "conformational" antibodies established synthetic ASPD for the 42-residue Aß(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. (13)C 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 ß-sheets. These results provide profound insight into the native ASPD, indicating that Aß is likely to self-assemble into the toxic intermediate with ß-sheet structures in AD brains. This approach can be applied to various intermediates relevant to amyloid diseases.

Aß(1-42) fibril structure illuminates self-recognition and replication of amyloid in Alzheimer's disease.

Increasing evidence has suggested that formation and propagation of misfolded aggregates of 42-residue human amyloid ß (Aß(1-42)), rather than of the more abundant Aß(1-40), provokes the Alzheimer's disease cascade. However, structural details of misfolded Aß(1-42) have remained elusive. Here we present the atomic model of an Aß(1-42) amyloid fibril, from solid-state NMR (ssNMR) data. It displays triple parallel-ß-sheet segments that differ from reported structures of Aß(1-40) fibrils. Remarkably, Aß(1-40) is incompatible with the triple-ß-motif, because seeding with Aß(1-42) fibrils does not promote conversion of monomeric Aß(1-40) into fibrils via cross-replication. ssNMR experiments suggest that C-terminal Ala42, absent in Aß(1-40), forms a salt bridge with Lys28 to create a self-recognition molecular switch that excludes Aß(1-40). The results provide insight into the Aß(1-42)-selective self-replicating amyloid-propagation machinery in early-stage Alzheimer's disease.

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.

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. et al., Nature 440, 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.

Capturing a reactive state of amyloid aggregates: NMR-based characterization of copper-bound Alzheimer disease amyloid ß-fibrils in a redox cycle.

The interaction of redox-active copper ions with misfolded amyloid ß (Aß) is linked to production of reactive oxygen species (ROS), which has been associated with oxidative stress and neuronal damages in Alzheimer disease. Despite intensive studies, it is still not conclusive how the interaction of Cu(+)/Cu(2+) with Aß aggregates leads to ROS production even at the in vitro level. In this study, we examined the interaction between Cu(+)/Cu(2+) and Aß fibrils by solid-state NMR (SSNMR) and other spectroscopic methods. Our photometric studies confirmed the production of ~60 µM hydrogen peroxide (H2O2) from a solution of 20 µM Cu(2+) ions in complex with Aß(1-40) in fibrils ([Cu(2+)]/[Aß] = 0.4) within 2 h of incubation after addition of biological reducing agent ascorbate at the physiological concentration (~1 mM). Furthermore, SSNMR (1)H T1 measurements demonstrated that during ROS production the conversion of paramagnetic Cu(2+) into diamagnetic Cu(+) occurs while the reactive Cu(+) ions remain bound to the amyloid fibrils. The results also suggest that O2 is required for rapid recycling of Cu(+) bound to Aß back to Cu(2+), which allows for continuous production of H2O2. Both (13)C and (15)N SSNMR results show that Cu(+) coordinates to Aß(1-40) fibrils primarily through the side chain Nδ of both His-13 and His-14, suggesting major rearrangements from the Cu(2+) coordination via Nε in the redox cycle. (13)C SSNMR chemical shift analysis suggests that the overall Aß conformations are largely unaffected by Cu(+) binding. These results present crucial site-specific evidence of how the full-length Aß in amyloid fibrils offers catalytic Cu(+) centers.

Sensitivity and resolution enhanced solid-state NMR for paramagnetic systems and biomolecules under very fast magic angle spinning.

Recent research in fast magic angle spinning (MAS) methods has drastically improved 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 (13)C and (1)H 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 least 20 kHz allows us to overcome major difficulties in (1)H and (13)C 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 (1)H T1 values, we can perform (1)H 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 (13)C biomolecular SSNMR measurements. Low-power (1)H decoupling schemes under VFMAS offer excellent spectral resolution for (13)C SSNMR by nominal (1)H RF irradiation at ∼10 kHz. By combining the VFMAS approach with enhanced (1)H T1 relaxation by paramagnetic doping, we can achieve extremely fast recycling in modern biomolecular SSNMR experiments. Experiments with (13)C-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 (13)C SSNMR measurements for biomolecules under faster MAS at a higher field. Our preliminary (13)C SSNMR data of Aß amyloid fibrils and GB1 microcrystals acquired at (1)H 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 (13)C SSNMR under ultrafast MAS at the spinning speed of ∼100 kHz.

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

Cu(2+) binding to Alzheimer's ß (Aß) 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 Aß 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 Aß are still largely unknown for Aß fibrils or other aggregates of Aß. 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 Aß(1-40). Selective quenching observed in (13)C SSNMR of Cu(2+)-bound Aß(1-40) suggested that primary Cu(2+) binding sites in Aß(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+)-Aß 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 Aß fibril, which are realized by both intra- and intermolecular contacts and highly concentrated coordination sites due to the in-register parallel ß-sheet arrangements.

Nanomole-scale protein solid-state NMR by breaking intrinsic 1HT1 boundaries.

We present an approach that accelerates protein solid-state NMR 5-20-fold using paramagnetic doping to condense data-collection time (to approximately 0.2 s per scan), overcoming a long-standing limitation on slow recycling owing to intrinsic (1)H T(1) longitudinal spin relaxation. Using low-power schemes under magic-angle spinning at 40 kHz, we obtained two-dimensional (13)C-(13)C and (13)C-(15)N solid-state NMR spectra for several to tens of nanomoles of beta-amyloid fibrils and ubiquitin in 1-2 d.