DNP-assisted solid-state NMR enables detection of proteins at nanomolar concentrations in fully protonated cellular milieu.

With the sensitivity enhancements conferred by dynamic nuclear polarization (DNP), magic angle spinning (MAS) solid state NMR spectroscopy experiments can attain the necessary sensitivity to detect very low concentrations of proteins. This potentially enables structural investigations of proteins at their endogenous levels in their biological contexts where their native stoichiometries with potential interactors is maintained. Yet, even with DNP, experiments are still sensitivity limited. Moreover, when an isotopically-enriched target protein is present at physiological levels, which typically range from low micromolar to nanomolar concentrations, the isotope content from the natural abundance isotopes in the cellular milieu can outnumber the isotope content of the target protein. Using isotopically enriched yeast prion protein, Sup35NM, diluted into natural abundance yeast lysates, we optimized sample composition. We found that modest cryoprotectant concentrations and fully protonated environments support efficient DNP. We experimentally validated theoretical calculations of the limit of specificity for an isotopically enriched protein in natural abundance cellular milieu. We establish that, using pulse sequences that are selective for adjacent NMR-active nuclei, proteins can be specifically detected in cellular milieu at concentrations in the hundreds of nanomolar. Finally, we find that maintaining native stoichiometries of the protein of interest to the components of the cellular environment may be important for proteins that make specific interactions with cellular constituents.

Spatially resolved DNP-assisted NMR illuminates the conformational ensemble of α-synuclein in intact viable cells.

The protein α-syn adopts a wide variety of conformations including an intrinsically disordered monomeric form and an α-helical rich membrane-associated form that is thought to play an important role in cellular membrane processes. However, despite the high affinity of α-syn for membranes, evidence that the α-helical form of α-syn is adopted inside cells has thus far been indirect. In cell DNP-assisted solid state NMR on frozen samples has the potential to report directly on the entire conformational ensemble. Moreover, because the DNP polarization agent can be dispersed both homogenously and inhomogenously throughout the cellular biomass, in cell DNP-assisted solid state NMR experiments can report either quantitatively upon the structural ensemble or can preferentially report upon the structural ensemble with a spatial bias. Using DNP-assisted MAS NMR we establish that the spectra of purified α-syn in the membrane-associated and intrinsically disordered forms have distinguishable spectra. When the polarization agent is introduced into cells by electroporation and dispersed homogenously, a minority of the α-syn inside HEK293 cells adopts a highly α-helical rich conformation. Alteration of the spatial distribution of the polarization agent preferentially enhances the signal from molecules nearer to the cellular periphery, thus the α-helical rich population is preferentially adopted toward the cellular periphery. This demonstrates how selectively altering the spatial distribution of the DNP polarization agent can be a powerful tool for preferential reporting on specific structural ensembles, paving the way for more nuanced investigations into the conformations that proteins adopt in different areas of the cell.

Conformational ensembles explain NMR spectra of frozen intrinsically disordered proteins.

Protein regions which are intrinsically disordered, exist as an ensemble of rapidly interconverting structures. Cooling proteins to cryogenic temperatures for dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR studies suspends most of the motions, resulting in peaks that are broad but not featureless. To demonstrate that detailed conformational restraints can be retrieved from the peak shapes of frozen proteins alone, we developed and used a simulation framework to assign peak features to conformers in the ensemble. We validated our simulations by comparing them to spectra of α-synuclein acquired under different experimental conditions. Our assignments of peaks to discrete dihedral angle populations suggest that structural constraints are attainable under cryogenic conditions. The ability to infer ensemble populations from peak shapes has important implications for DNP MAS NMR studies of proteins with regions of disorder in living cells because chemical shifts are the most accessible measured parameter.

Stability of the nitroxide biradical AMUPol in intact and lysed mammalian cells.

Dynamic Nuclear Polarization (DNP) enhanced solid state NMR increases experimental sensitivity, potentially enabling detection of biomolecules at their physiological concentrations. The sensitivity of DNP experiments is due to the transfer of polarization from electron spins of free radicals to the nuclear spins of interest. Here, we investigate the reduction of AMUPol in both lysed and intact HEK293 cells. We find that nitroxide radicals are reduced with first order reduction kinetics by cell lysates at a rate of âˆ¼ 12% of the added nitroxide radical concentration per hour. We also found that electroporation delivered a consistent amount of AMUPol to intact cells and that nitroxide radicals are reduced just slightly more rapidly (∼15% per hour) by intact cells than by cell lysates. The two nitroxide radicals of AMUPol are reduced independently and this leads to considerable accumulation of the DNP-silent monoradical form of AMUPol, particularly in preparations of intact cells where nearly half of the AMUPol is already reduced to the DNP silent monoradical form at the earliest experimental time points. This confirms that the loss of the DNP-active biradical form of AMUPol is faster than the nitroxide reduction rate. Finally, we investigate the effect of adding N-ethyl maleimide, a well-known inhibitor of thiol (-SH) group-based reduction of nitroxide biradicals in cells, on AMUPol reduction, cellular viability, and DNP performance. Although pre-treatment of cells with NEM effectively inhibited the reduction of AMUPol, exposure to NEM compromised cellular viability and, surprisingly, did not improve DNP performance. Collectively, these results indicate that, currently, the most effective strategy to obtain high DNP enhancements for DNP-assisted in-cell NMR is to minimize room temperature contact times with cellular constituents and suggest that the development of bio-resistant polarization agents for DNP could considerably increase the sensitivity of DNP-assisted in-cell NMR experiments.

In-Cell Sensitivity-Enhanced NMR of Intact Viable Mammalian Cells.

NMR has the resolution and specificity to determine atomic-level protein structures of isotopically labeled proteins in complex environments, and with the sensitivity gains conferred by dynamic nuclear polarization (DNP), NMR has the sensitivity to detect proteins at their endogenous concentrations. However, DNP sensitivity enhancements are critically dependent on experimental conditions and sample composition. While some of these conditions are theoretically compatible with cellular viability, the effects of others on cellular sample integrity are unknown. Uncertainty about the integrity of cellular samples limits the utility of experimental outputs of in-cell experiments. Using several measures, we establish conditions that support DNP enhancements that can enable detection of micromolar concentrations of proteins in experimentally tractable times that are compatible with cellular viability. Taken together, we establish DNP-assisted MAS NMR as a technique for structural investigations of biomolecules in intact viable cells that can be phenotyped both before and after NMR experiments.

Sensitivity-Enhanced Solid-State NMR Detection of Structural Differences and Unique Polymorphs in Pico- to Nanomolar Amounts of Brain-Derived and Synthetic 42-Residue Amyloid-ß Fibrils.

Amyloid-ß (Aß) fibrils in neuritic plaques are a hallmark of Alzheimer's disease (AD). Since the 42-residue Aß (Aß42) fibril is the most pathogenic among different Aß species, its structural characterization is crucial to our understanding of AD. While several polymorphs have been reported for Aß40, previous studies of Aß42 fibrils prepared at neutral pH detected essentially only one structure, with an S-shaped ß-sheet arrangement (e.g., Xiao et al. Nat. Struct. Mol. Biol. 2015, 22, 499). Herein, we demonstrate the feasibility of characterizing the structure of trace amounts of brain-derived and synthetic amyloid fibrils by sensitivity-enhanced 1H-detected solid-state NMR (SSNMR) under ultrafast magic angle spinning. By taking advantage of the high sensitivity of this technique, we first demonstrate its applicability for the high-throughput screening of trace amounts of selectively 13C- and 15N-labeled Aß42 fibril prepared with ∼0.01% patient-derived amyloid (ca. 4 pmol) as a seed. The comparison of 2D 13C/1H SSNMR data revealed marked structural differences between AD-derived Aß42 (∼40 nmol or ∼200 µg) and synthetic fibrils in less than 10 min, confirming the feasibility of assessing the fibril structure from ∼1 pmol of brain amyloid seed in ∼2.5 h. We also present the first structural characterization of synthetic fully protonated Aß42 fibril by 1H-detected 3D and 4D SSNMR. With procedures assisted by automated assignments, main-chain resonance assignments were completed for trace amounts (∼42 nmol) of a fully protonated amyloid fibril in the 1H-detection approach. The results suggest that this Aß42 fibril exhibits a novel fold or polymorph structure.

In-Cell NMR of Intact Mammalian Cells Preserved with the Cryoprotectants DMSO and Glycerol Have Similar DNP Performance.

NMR has the resolution and specificity to determine atomic-level protein structures of isotopically-labeled proteins in complex environments and, with the sensitivity gains conferred by dynamic nuclear polarization (DNP), NMR has the sensitivity to detect proteins at their endogenous concentrations. Prior work established that DNP MAS NMR is compatible with cellular viability. However, in that work, 15% glycerol, rather than the more commonly used 10% DMSO, was used as the cellular cryoprotectant. Moreover, incubation of cells cryoprotected 15% glycerol with the polarization agent, AMUPol, resulted in an inhomogeneous distribution of AMUPol through the cellular biomass, which resulted in a spatial bias of the NMR peak intensities. Because 10% DMSO is not only the most used cryoprotectant for mammalian cells, but also because DMSO is often used to improve delivery of molecules to cells, we sought to characterize the DNP performance of cells that were incubated with AMUPol and cryoprotected with 10% DMSO. We found that, like cells preserved with 15% glycerol, cells preserved with 10% DMSO retain high viability during DNP MAS NMR experiments if they are frozen at a controlled rate. However, DMSO did not improve the dispersion of AMUPol throughout the cellular biomass. Cells preserved with 15% glycerol and with 10% DMSO had similar DNP performance for both the maximal DNP enhancements as well as the inhomogeneous dispersion of AMUPol throughout the cellular biomass. Therefore, 10% DMSO and 15% glycerol are both appropriate cryoprotectant systems for DNP-assisted MAS NMR of intact viable mammalian cells.

Michler's hydrol blue elucidates structural differences in prion strains.

Yeast prions provide self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that confer distinct phenotypes when introduced into cells that do not carry the prion. Classic dyes, such as thioflavin T and Congo red, exhibit large increases in fluorescence when bound to amyloids, but these dyes are not sensitive to local structural differences that distinguish amyloid strains. Here we describe the use of Michler's hydrol blue (MHB) to investigate fibrils formed by the weak and strong prion fibrils of Sup35NM and find that MHB differentiates between these two polymorphs. Quantum mechanical time-dependent density functional theory (TDDFT) calculations indicate that the fluorescence properties of amyloid-bound MHB can be correlated to the change of binding site polarity and that a tyrosine to phenylalanine substitution at a binding site could be detected. Through the use of site-specific mutants, we demonstrate that MHB is a site-specific environmentally sensitive probe that can provide structural details about amyloid fibrils and their polymorphs.

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability.

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.

NMR-based site-resolved profiling of ß-amyloid misfolding reveals structural transitions from pathologically relevant spherical oligomer to fibril.

Increasing evidence highlights the central role of neurotoxic oligomers of the 42-residue-long ß-amyloid (Aß42) in Alzheimer's disease (AD). However, very limited information is available on the structural transition from oligomer to fibril, particularly for pathologically relevant amyloids. To the best of our knowledge, we present here the first site-specific structural characterization of Aß42 misfolding, from toxic oligomeric assembly yielding a similar conformation to an AD-associated Aß42 oligomer, into a fibril. Transmission EM (TEM) analysis revealed that a spherical amyloid assembly (SPA) of Aß42 with a 15.6 ± 2.1-nm diameter forms in a ∼30-µm Aß42 solution after a ∼10-h incubation at 4 °C, followed by a slow conversion into fibril at ∼180 h. Immunological analysis suggested that the SPA has a surface structure similar to that of amylospheroid (ASPD), a patient-derived toxic Aß oligomer, which had a diameter of 10-15 nm in negative-stain TEM. Solid-state NMR analyses indicated that the SPA structure involves a ß-loop-ß motif, which significantly differed from the triple-ß motif observed for the Aß42 fibril. The comparison of the 13C chemical shifts of SPA with those of the fibril prepared in the above conditions and interstrand distance measurements suggested a large conformational change involving rearrangements of intermolecular ß-sheet into in-register parallel ß-sheet during the misfolding. A comparison of the SPA and ASPD 13C chemical shifts indicated that SPA is structurally similar to the ASPD relevant to AD. These observations provide insights into the architecture and key structural transitions of amyloid oligomers relevant for AD pathology.

DNP-Assisted NMR Investigation of Proteins at Endogenous Levels in Cellular Milieu.

Structural investigations of biomolecules are typically confined to in vitro systems under extremely limited conditions. These investigations yield invaluable insights, but such experiments cannot capture important structural features imposed by cellular environments. Structural studies of proteins in their native contexts are not only possible using state-of-the-art sensitivity-enhanced (dynamic nuclear polarization, DNP) solid-state nuclear magnetic resonance (NMR) techniques, but these studies also demonstrate that the cellular context can and does have a dramatic influence on protein structure. In this chapter, we describe methods to prepare samples of isotopically labeled proteins at endogenous levels in cellular contexts alongside quality control methods to ensure that such samples accurately model important features of the cellular environment.

Solid-State NMR Studies of Amyloid Materials: A Protocol to Define an Atomic Model of Aß(1-42) in Amyloid Fibrils.

Intense efforts have been made to understand the molecular structures of misfolded amyloid ß (Aß) in order to gain insight into the pathological mechanism of Alzheimer's disease. Solid-state NMR spectroscopy (SSNMR) is considered a primary tool for elucidating the structures of insoluble and noncrystalline amyloid fibrils and other amyloid assemblies. In this chapter, we describe a detailed protocol to obtain the first atomic model of the 42-residue human Aß peptide Aß(1-42) in structurally homogeneous amyloid fibrils from our recent SSNMR study (Nat Struct Mol Biol 22:499-505, 2015). Despite great biological and clinical interest in Aß(1-42) fibrils, their structural details have been long-elusive until this study. The protocol is divided into four sections. First, the solid-phase peptide synthesis (SPPS) and purification of monomeric Aß(1-42) is described. We illustrate a controlled incubation method to prompt misfolding of Aß(1-42) into homogeneous amyloid fibrils in an aqueous solution with fragmented Aß(1-42) fibrils as seeds. Next, we detail analysis of Aß(1-42) fibrils by SSNMR to obtain structural restraints. Finally, we describe methods to construct atomic models of Aß(1-42) fibrils based on SSNMR results through two-stage molecular dynamics calculations.

E22G Pathogenic Mutation of ß-Amyloid (Aß) Enhances Misfolding of Aß40 by Unexpected Prion-like Cross Talk between Aß42 and Aß40.

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 ß-amyloid (Aß) is widely considered to trigger amyloid plaque deposition. Despite increasing evidence that misfolded Aß mimics prions, interactions of misfolded 42-residue Aß42 with more abundant 40-residue Aß40 in AD are elusive. This study presents in vitro evidence that a heterozygous E22G pathogenic ("Arctic") mutation of Aß40 can enhance misfolding of Aß via cross-seeding from wild-type (WT) Aß42 fibril. Thioflavin T (ThT) fluorescence analysis suggested that misfolding of E22G Aß40 was enhanced by adding 5% (w/w) WT Aß42 fibril as "seed", whereas WT Aß40 was unaffected by Aß42 fibril seed. 13C SSNMR analysis revealed that such cross-seeding prompted formation of E22G Aß40 fibril that structurally mimics the seed Aß42 fibril, suggesting unexpected cross talk of Aß 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.

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.

Simultaneous discrimination of jasmonic acid stereoisomers by CE-QTOF-MS employing the partial filling technique.

Jasmonic acid (JA), an essential plant hormone controlling the plant defense signaling system and developmental processes, has stereospecific bioactivities that have not been well understood mainly due to the limitation in separation and detection methodologies. In this work, a fast CE-UV method based on short-end injection technique and a sensitive CE-QTOF-MS method based on partial filling technique were successfully developed for the enantioseparation of racemic JA. The successive coating technique was also involved by modifying the capillary with multiple ionic polymer layers of polybrene-dextran sulfate-polybrene. This was the first report on the direct resolution of both pairs of JA enantiomers, including two naturally occurring JA stereoisomers. Although no pure JA stereoisomers were commercially available, all the separated JA stereoisomers were identified indirectly by comparing the difference between the racemic standard and plant samples based on the presence and the ratio of each stereoisomer. Satisfactory results were obtained in terms of sensitivity (LOD, 24 ng/mL or 0.7 fmol for single JA stereoisomer) using 45 mmol/L ammonium acetate at pH 4.5 containing 70 mmol/L α-CD as the buffer system. This established CE-QTOF-MS method was later successfully applied for the study of the naturally occurring JA stereoisomers in wounded tobacco leaves.

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.