Revealing Protein Structures in Solid-Phase Peptide Synthesis by ¹³C Solid-State NMR: Evidence of Excessive Misfolding for Alzheimer’s β

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

E22G Pathogenic Mutation of β‑Amyloid (Aβ) Enhances Misfolding of Aβ40 by Unexpected Prion-like Cross Talk between Aβ42 and Aβ40

Cross-seeding of misfolded amyloid proteins is postulated to induce cross-species infection of prion diseases. In sporadic Alzheimer’s disease (AD), misfolding of 42-residue β-amyloid (Aβ) is widely considered to trigger amyloid plaque deposition. Despite increasing evidence that misfolded Aβ mimics prions, interactions of misfolded 42-residue Aβ42 with more abundant 40-residue Aβ40 in AD are elusive. This study presents <i>in vitro</i> evidence that a heterozygous E22G pathogenic (“Arctic”) mutation of Aβ40 can enhance misfolding of Aβ via cross-seeding from wild-type (WT) Aβ42 fibril. Thioflavin T (ThT) fluorescence analysis suggested that misfolding of E22G Aβ40 was enhanced by adding 5% (w/w) WT Aβ42 fibril as “seed”, whereas WT Aβ40 was unaffected by Aβ42 fibril seed. ¹³C SSNMR analysis revealed that such cross-seeding prompted formation of E22G Aβ40 fibril that structurally mimics the seed Aβ42 fibril, suggesting unexpected cross talk of Aβ isoforms that potentially promotes early onset of AD. The SSNMR approach is likely applicable to elucidate structural details of heterogeneous amyloid fibrils produced in cross-seeding for amyloids linked to neurodegenerative diseases

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

Synthesis of ¹³C‑,¹⁵N‑Labeled Graphitic Carbon Nitrides and NMR-Based Evidence of Hydrogen-Bonding Assisted Two-Dimensional Assembly

Graphitic carbon nitride (g-C₃N₄) has gained great attention as a material of promise for artificial photosynthesis. In place of synthesis of traditional three-dimensional g-C₃N₄ via polymerization of melamine or melem, recent studies seek to establish an alternative synthetic approach for two-dimensional g-C₃N₄ using a smaller precursor such as urea. However, the effectiveness of such a synthetic approach and resultant polymeric forms of g-C₃N₄ in this approach are still largely unknown. In this study, we present that solid-state NMR (SSNMR) analysis for ¹³C- and ¹⁵N-labeled g-C₃N₄ prepared from urea offers an unparalleled structural view for the heterogeneous in-plane structure of g-C₃N₄ and most likely for its moieties. We revealed that urea was successfully assembled in melem oligomers, which include extended oligomers involving six or more melem subunits. SSNMR, transmission electron micrograph, and <i>ab initio</i> calculation data suggested that the melem oligomer units were further extended into graphene-like layered materials via widespread NH–N hydrogen bonds between oligomers

NMR-Based Structural Modeling of Graphite Oxide Using Multidimensional ¹³C Solid-State NMR and ab Initio Chemical Shift Calculations

Chemically modified graphenes and other graphite-based materials have attracted growing interest for their unique potential as lightweight electronic and structural nanomaterials. It is an important challenge to construct structural models of noncrystalline graphite-based materials on the basis of NMR or other spectroscopic data. To address this challenge, a solid-state NMR (SSNMR)-based structural modeling approach is presented on graphite oxide (GO), which is a prominent precursor and interesting benchmark system of modified graphene. An experimental 2D ¹³C double-quantum/single-quantum correlation SSNMR spectrum of ¹³C-labeled GO was compared with spectra simulated for different structural models using ab initio geometry optimization and chemical shift calculations. The results show that the spectral features of the GO sample are best reproduced by a geometry-optimized structural model that is based on the Lerf−Klinowski model (Lerf, A. et al. <i>Phys. Chem. B</i> <b>1998</b>, <i>102</i>, 4477); this model is composed of interconnected sp², 1,2-epoxide, and COH carbons. This study also convincingly excludes the possibility of other previously proposed models, including the highly oxidized structures involving 1,3-epoxide carbons (Szabo, I. et al. <i>Chem. Mater.</i> <b>2006</b>, <i>18</i>, 2740). ¹³C chemical shift anisotropy (CSA) patterns measured by a 2D ¹³C CSA/isotropic shift correlation SSNMR were well reproduced by the chemical shift tensor obtained by the ab initio calculation for the former model. The approach presented here is likely to be applicable to other chemically modified graphenes and graphite-based systems

Progress in 13C and 1H solid-state nuclear magnetic resonance for paramagnetic systems under very fast magic angle spinning.

High-resolution solid-state NMR (SSNMR) of paramagnetic systems has been largely unexplored because of various technical difficulties due to large hyperfine shifts, which have limited the success of previous studies through depressed sensitivity/resolution and lack of suitable assignment methods. Our group recently introduced an approach using "very fast" magic angle spinning (VFMAS) for SSNMR of paramagnetic systems, which opened an avenue toward routine analyses of small paramagnetic systems by (13)C and (1)H SSNMR [Y. Ishii et al., J. Am. Chem. Soc. 125, 3438 (2003); N. P. Wickramasinghe et al., ibid. 127, 5796 (2005)]. In this review, we discuss our recent progress in establishing this approach, which offers solutions to a series of problems associated with large hyperfine shifts. First, we demonstrate that MAS at a spinning speed of 20 kHz or higher greatly improves sensitivity and resolution in both (1)H and (13)C SSNMR for paramagnetic systems such as Cu(II)(DL-alanine)(2)H(2)O (Cu(DL-Ala)(2)) and Mn(acac)(3), for which the spectral dispersions due to (1)H hyperfine shifts reach 200 and 700 ppm, respectively. Then, we introduce polarization transfer methods from (1)H spins to (13)C spins with high-power cross polarization and dipolar insensitive nuclei enhanced by polarization transfer (INEPT) in order to attain further sensitivity enhancement and to correlate (1)H and (13)C spins in two-dimensional (2D) SSNMR for the paramagnetic systems. Comparison of (13)C VFMAS SSNMR spectra with (13)C solution NMR spectra revealed superior sensitivity in SSNMR for Cu(DL-Ala)(2), Cu(Gly)(2), and V(acac)(3). We discuss signal assignment methods using one-dimensional (1D) (13)C SSNMR (13)C-(1)H rotational echo double resonance (REDOR) and dipolar INEPT methods and 2D (13)C(1)H correlation SSNMR under VFMAS, which yield reliable assignments of (1)H and (13)C resonances for Cu(Ala-Thr). Based on the excellent sensitivity/resolution and signal assignments attained in the VFMAS approach, we discuss methods of elucidating multiple distance constraints in unlabeled paramagnetic systems by combing simple measurements of (13)C T(1) values and anisotropic hyperfine shifts. Comparison of experimental (13)C hyperfine shifts and ab initio calculated shifts for alpha- and beta-forms of Cu(8-quinolinol)(2) demonstrates that (13)C hyperfine shifts are parameters exceptionally sensitive to small structural difference between the two polymorphs. Finally, we discuss sensitivity enhancement with paramagnetic ion doping in (13)C SSNMR of nonparamagnetic proteins in microcrystals. Fast recycling with exceptionally short recycle delays matched to short (1)H T(1) of approximately 60 ms in the presence of Cu(II) doping accelerated 1D (13)C SSNMR for ubiquitin and lysozyme by a factor of 7.3-8.4 under fast MAS at a spinning speed of 40 kHz. It is likely that the VFMAS approach and use of paramagnetic interactions are applicable to a variety of paramagnetic systems and nonparamagnetic biomolecules

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

Structure–Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of <i>MYLK</i> Variants

<div><p>The <i>MYLK</i> gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three <i>MYLK</i> SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by ¹H-¹⁵N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by <i>in silico</i> molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these <i>MYLK</i> SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm <i>MYLK</i> as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.</p></div