RESUMO
A complex interplay between various processes underlies the neuropathology of Alzheimer's disease (AD) and its progressive course. Several lines of evidence point to the coupling between Aß aggregation and neuroinflammation and its role in maintaining brain homeostasis during the long prodromal phase of AD. Little is however known about how this protective mechanism fails and as a result, an irreversible and progressive transition to clinical AD occurs. Here, we introduce a minimal model of a coupled system of Aß aggregation and inflammation, numerically simulate its dynamical behavior, and analyze its bifurcation properties. The introduced model represents the following events: generation of Aß monomers, aggregation of Aß monomers into oligomers and fibrils, induction of inflammation by Aß aggregates, and clearance of various Aß species. Crucially, the rates of Aß generation and clearance are modulated by inflammation level following a Hill-type response function. Despite its relative simplicity, the model exhibits enormously rich dynamics ranging from overdamped kinetics to sustained oscillations. We then specify the region of inflammation- and coupling-related parameters space where a transition to oscillatory dynamics occurs and demonstrate how changes in Aß aggregation parameters could shift this oscillatory region in parameter space. Our results reveal the propensity of coupled Aß aggregation-inflammation systems to oscillatory dynamics and propose prolonged sustained oscillations and their consequent immune system exhaustion as a potential mechanism underlying the transition to a more progressive phase of amyloid pathology in AD. The implications of our results in regard to early diagnosis of AD and anti-AD drug development are discussed.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Inflamação , Peptídeos beta-Amiloides/metabolismo , Humanos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Inflamação/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Biológicos , Agregação Patológica de Proteínas/metabolismo , Agregados ProteicosRESUMO
Metal ions, including biologically prevalent sodium ions, can modulate electrostatic interactions frequently involved in the stability of condensed compartments in cells. Quantitative characterization of heterogeneous ion dynamics inside biomolecular condensates demands new experimental approaches. Here we develop a 23Na NMR relaxation-based integrative approach to probe dynamics of sodium ions inside agarose gels as a model system. We exploit the electric quadrupole moment of spin-3/2 23Na nuclei and, through combination of single-quantum and triple-quantum-filtered 23Na NMR relaxation methods, disentangle the relaxation contribution of different populations of sodium ions inside gels. Three populations of sodium ions are identified: a population with bi-exponential relaxation representing ions within the slow motion regime and two populations with mono-exponential relaxation but at different rates. Our study demonstrates the dynamical heterogeneity of sodium ions inside agarose gels and presents a new experimental approach for monitoring dynamics of sodium and other spin-3/2 ions (e.g. chloride) in condensed environments.
Assuntos
Géis , Sefarose , Sódio , Sefarose/química , Sódio/química , Géis/química , Espectroscopia de Ressonância Magnética/métodos , Íons/química , Teoria QuânticaRESUMO
Liquid-liquid phase separation is the key process underlying formation of membrane-less compartments in cells. A highly dynamic cellular body with rapid component exchange is Cajal body (CB), which supports the extensive compositional dynamics of the RNA splicing machinery, spliceosome. Here, we select an arginine-glycine (RG)-rich segment of coilin, the major component of CB, establish its RNA-induced phase separation, and through combined use of nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) probes, interrogate its dynamics within the crowded interior of formed droplets. Taking advantage of glycine-based singlet-states, we show that glycines retain a large level of sub-nanoseconds dynamics inside the coilin droplets. Furthermore, the continuous-wave (CW) and electron-electron dipolar (PELDOR) and electron-nucleus hyperfine coupling EPR data (HYSCORE) support the RNA-induced formation of dynamic coilin droplets with high coilin peptide concentrations. The combined NMR and EPR data reveal the high dynamics of the RG-rich coilin within droplets and suggest its potential role in the large dynamics of CBs.
Assuntos
Arginina , Proteínas Nucleares , Proteínas Nucleares/genética , Glicina , Elétrons , RNA , Corpos EnoveladosRESUMO
Signal transduction by the ligated B cell antigen receptor (BCR) depends on the preorganization of its intracellular components, such as the effector proteins SLP65 and CIN85 within phase-separated condensates. These liquid-like condensates are based on the interaction between three Src homology 3 (SH3) domains and the corresponding proline-rich recognition motifs (PRM) in CIN85 and SLP65, respectively. However, detailed information on the protein conformation and how it impacts the capability of SLP65/CIN85 condensates to orchestrate BCR signal transduction is still lacking. This study identifies a hitherto unknown intramolecular SH3:PRM interaction between the C-terminal SH3 domain (SH3C) of CIN85 and an adjacent PRM. We used high-resolution nuclear magnetic resonance (NMR) experiments to study the flexible linker region containing the PRM and determined the extent of the interaction in multidomain constructs of the protein. Moreover, we observed that the phosphorylation of a serine residue located in the immediate vicinity of the PRM regulates this intramolecular interaction. This allows for a dynamic modulation of CIN85's valency toward SLP65. B cell culture experiments further revealed that the PRM/SH3C interaction is crucial for maintaining the physiological level of SLP65/CIN85 condensate formation, activation-induced membrane recruitment of CIN85, and subsequent mobilization of Ca2+. Our findings therefore suggest that the intramolecular interaction with the adjacent disordered linker is effective in modulating CIN85's valency both in vitro and in vivo. This therefore constitutes a powerful way for the modulation of SLP65/CIN85 condensate formation and subsequent B cell signaling processes within the cell.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/química , Transdução de Sinais/fisiologia , Domínios de Homologia de src , Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Ligação ProteicaRESUMO
Liquid water is essential for life as we know it and the coupling between water and biomolecular dynamics is crucial for life processes. Jupiter's moon Europa is a good candidate for searching for extraterrestrial life in our outer solar system, mainly because a liquid water salty ocean in contact with a rocky seafloor underlies its ice shell. Little, however, is known about the chemical composition of the subglacial ocean of Europa or the brine pockets within its ice shell and their impacts on water dynamics. Here, we employ 1H, 17O, 23Na and 35Cl NMR spectroscopy, especially NMR spin relaxation and diffusion methods, and investigate the mobility of water molecules and ions in eutectic solutions of magnesium sulfate and sodium chloride, two salts ubiquitously present on the surface of Europa, over a range of temperatures and pressures pertinent to Europa's subglacial ocean. The NMR data demonstrate the more pronounced effect of magnesium sulfate compared with sodium chloride on the mobility of water molecules. Even at its much lower eutectic temperature, the sodium chloride solution retains a relatively large level of water mobility. Our results highlight the higher potential of a sodium chloride-rich than magnesium sulfate-rich Europa's ocean to accommodate life and support life origination within the eutectic melts of Europa's ice shell.
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Little is known about how maturation of Alzheimer's disease-related amyloid ß (Aß) fibrils alters their stability and potentially influences their spreading in the brain. Using high-pressure NMR, we show that progression from early to late Aß40 aggregates enhances the kinetic stability, while ageing during weeks to months enhances their thermodynamic stability.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Humanos , Peptídeos beta-Amiloides/química , Espectroscopia de Ressonância Magnética , Amiloide/química , Fragmentos de Peptídeos/químicaRESUMO
Amyloid-beta (Aß) deposition as senile plaques is a pathological hallmark of Alzheimer's disease (AD). AD is characterized by a large level of heterogeneity in amyloid pathology, whose molecular origin is poorly understood. Here, we employ NMR spectroscopy and MD simulation at ambient and high pressures and investigate how AD-related mutations in Aß peptide influence the stability of Aß aggregates. The pressure-induced monomer dissociation from Aß aggregates monitored by NMR demonstrated that the Iowa (D23N), Arctic (E22G), and Osaka (ΔE22) mutations altered the pressure stability of Aß40 aggregates in distinct manners. While the NMR data of monomeric Aß40 showed only small localized effects of mutations, the MD simulation of mutated Aß fibrils revealed their distinct susceptibility to elevated pressure. Our data propose a structural basis for the distinct stability of various Aß fibrils and highlights "stability" as a molecular property potentially contributing to the large heterogeneity of amyloid pathology in AD.
Assuntos
Doença de Alzheimer , Humanos , Doença de Alzheimer/genética , Amiloide/química , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/química , Espectroscopia de Ressonância Magnética , Mutação , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/químicaRESUMO
Reorientational dynamics of intrinsically disordered proteins (IDPs) contain multiple motions often clustered around three motional modes: ultrafast librational motions of amide groups, fast local backbone conformational fluctuations and slow chain segmental motions. This dynamic picture is mainly based on 15 Nâ NMR relaxation studies of IDPs at relatively low temperatures where the amide-water proton exchange rates are sufficiently small. Less is known, however, about the dynamics of IDPs at more physiological temperatures. Here, we investigate protein dynamics in a 441-residue long IDP, tau protein, in the temperature range from 0-25 °C, using 15 Nâ NMR relaxation rates and spectral density analysis. While at these temperatures relaxation rates are still better described in terms of amide group librational motions, local backbone dynamics and chain segmental motions, the temperature-dependent trend of spectral densities suggests that the timescales of fast backbone conformational fluctuations and slower chain segmental motions might become inseparable at higher temperatures. Our data demonstrate the remarkable dynamic plasticity of this prototypical IDP and highlight the need for dynamic studies of IDPs at multiple temperatures.
Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas tau , Temperatura , Conformação Proteica , Espectroscopia de Ressonância Magnética , Proteínas Intrinsicamente Desordenadas/química , AmidasRESUMO
Heparins and heparan sulfate polysaccharides are negatively charged glycosaminoglycans and play important roles in cell-to-matrix and cell-to-cell signaling processes. Metal ion binding to heparins alters the conformation of heparins and influences their function. Various experimental techniques have been used to investigate metal ion-heparin interactions, frequently with inconsistent results. Exploiting the quadrupolar 23Na nucleus, we herein develop a 23Na NMR-based competition assay and monitor the binding of divalent Ca2+ and Mg2+ and trivalent Al3+ metal ions to sodium heparin and the consequent release of sodium ions from heparin. The 23Na spin relaxation rates and translational diffusion coefficients are utilized to quantify the metal ion-induced release of sodium ions from heparin. In the case of the Al3+ ion, the complementary approach of 27Al quadrupolar NMR is employed as a direct probe of ion binding to heparin. Our NMR results demonstrate at least two metal ion-binding sites with different affinities on heparin, potentially undergoing dynamic exchange. For the site with lower metal ion binding affinity, the order of Ca2+ > Mg2+ > Al3+ is obtained, in which even the weakly binding Al3+ ion is capable of displacing sodium ions from heparin. Overall, the multinuclear quadrupolar NMR approach employed here can monitor and quantify metal ion binding to heparin and capture different modes of metal ion-heparin binding.
Assuntos
Heparina , Heparitina Sulfato , Heparina/química , Espectroscopia de Ressonância Magnética/métodos , Heparitina Sulfato/metabolismo , Metais/metabolismo , Íons , Sódio/metabolismo , Sítios de LigaçãoRESUMO
Natural compounds with anti-aggregation capacity are increasingly recognized as viable candidates against neurodegenerative diseases. Recently, the polyphenolic fraction of propolis (PFP), a complex bee product, has been shown to inhibit amyloid aggregation of a model protein especially in the nanosheet form. Here, we examine the aggregation-modulating effects of the PFP nanosheets on α-synuclein (α-syn), an intrinsically disordered protein involved in the pathogenesis of Parkinson's disease. Based on a range of biophysical data including intrinsic and extrinsic fluorescence, circular dichroism (CD) data, and nuclear magnetic resonance spectroscopy, we propose a model for the interaction of α-syn with PFP nanosheets, where the positively charged N-terminal and the middle non-amyloid component regions of α-syn act as the main binding sites with the negatively charged PFP nanosheets. The Thioflavin T (ThT) fluorescence, Congo red absorbance, and CD data reveal a prominent dose-dependent inhibitory effect of PFP nanosheets on α-syn amyloid aggregation, and the microscopy images and MTT assay data suggest that the PFP nanosheets redirect α-syn aggregation toward nontoxic off-pathway oligomers. When preformed α-syn amyloid fibrils are present, fluorescence images show co-localization of PFP nanosheets and ThT, further confirming the binding of PFP nanosheets with α-syn amyloid fibrils. Taken together, our results demonstrate the binding and anti-aggregation activity of PFP nanosheets in a disease-related protein system and propose them as potential nature-based tools for probing and targeting pathological protein aggregates in neurodegenerative diseases.
Assuntos
Própole , alfa-Sinucleína , alfa-Sinucleína/metabolismo , Amiloide/metabolismo , Própole/farmacologia , Polifenóis/farmacologia , Agregados ProteicosRESUMO
In their Comment (DOI: 10.1021/jacs.2c02965) on two related publications by our group (J. Am. Chem. Soc. 2022, 144, 1380-1388; DOI: 10.1021/jacs.1c11754) and another (J. Am. Chem. Soc. 2021, 143, 20884-20890; DOI: 10.1021/jacs.1c09455), Huang and Granick refer to the diffusion NMR measurements of molecules during a copper-catalyzed azide-alkyne cycloaddition (CuAAC) "click" reaction. Here we respond to their comments and maintain that no measurable diffusion enhancement was observed during the reaction. We expand on the physical arguments presented in our original JACS Article regarding the appropriate reference state for the diffusion coefficient and present new data showing that the use of other reference states, as suggested by Huang and Granick, will still support our conclusion that the two reactants and one product of the CuAAC reaction do not exhibit boosted mobility during the reaction.
Assuntos
Azidas , Química Click , Alcinos/química , Azidas/química , Catálise , Cobre/química , Reação de CicloadiçãoRESUMO
Increasing the temperature by just a few degrees may lead to structural perturbation or unfolding of the protein and consequent loss of function. The concepts of flexibility and rigidity are fundamental for understanding the relationships between function, structure and stability. Protein unfolding can often be triggered by thermal fluctuations with flexible residues usually on the protein surface. Therefore, identification and knowledge of the effect of modification to flexible regions in protein structures are required for efficient protein engineering and the rational design of thermally stable proteins. The most flexible regions in protein are loops, hence their rigidification is one of the effective strategies for increasing thermal stability. Directed evolution or rational design by computational prediction can also lead to the generation of thermally stable proteins. Computational protein design has been improved significantly in recent years and has successfully produced de novo stable backbone structures with optimized sequences and functions. This review discusses intramolecular and intermolecular interactions that determine the protein structure, and the strategies utilized in the mutagenesis of mesophilic proteins to stabilize and improve the functional characteristics of biocatalysts by describing efficient techniques and strategies to rigidify flexible loops at appropriate positions in the structure of the protein.
Assuntos
Engenharia de Proteínas , Desdobramento de Proteína , Engenharia de Proteínas/métodos , Estabilidade Proteica , Proteínas/genética , TemperaturaRESUMO
Living cells often contain compartments with high concentration of charged biomolecules. A key question pertinent to the function of biomolecules is how water dynamics are affected by interaction with charged molecules. Here, we study the dynamical behavior of water in an extreme condition, that is, in saturated salt solutions, where nearly all water molecules are located within the first hydration layer of ions. To facilitate disentangling the effects of cations and anions, our study is focused on alkali chloride solutions. Following a multi-nuclear NMR approach enabling direct monitoring of protons and the quadrupolar nuclei 7 Li, 17 O, 23 Na, 35 Cl, 87 Rb and 133 Cs, we investigate how the translational and rotational mobility of water molecules, chloride anion and corresponding cations are affected within the constrained environment of saturated solutions. Our results indicate that water molecules preserve a large level of mobility within saturated alkali chloride solutions that is significantly independent of adjacent ions.
Assuntos
Cloretos , Água , Álcalis , Ânions/química , Cátions/química , Espectroscopia de Ressonância Magnética , Cloreto de Sódio , Cloreto de Sódio na Dieta , Água/químicaRESUMO
Biomolecular phase separation plays a key role in the spatial organization of cellular activities. Dynamic formation and rapid component exchange between phase separated cellular bodies and their environment are crucial for their function. Here, we employ a well-established phase separating model system, namely, a triethylamine (TEA)-water mixture, and develop an NMR approach to detect the exchange of scaffolding TEA molecules between separate phases and determine the underlying exchange rate. We further demonstrate how the advantageous NMR properties of fluorine nuclei provide access to otherwise inaccessible exchange processes of a client molecule. The developed NMR-based approach allows quantitative monitoring of the effect of regulatory factors on component exchange and facilitates "exchange"-based screening and optimization of small molecules against druggable biomolecular targets located inside condensed phases.
Assuntos
Flúor , Imageamento por Ressonância Magnética , Humanos , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Ressonância Magnética Nuclear BiomolecularRESUMO
Alzheimer's disease is characterized by the presence of extraneuronal amyloid plaques composed of amyloid-beta (Aß) fibrillar aggregates in the brains of patients. In mouse models, it has previously been shown that atorvastatin (Ator), a cholesterol-lowering drug, has some reducing effect on the production of cerebral Aß. A meta-analysis on humans showed moderate effects in the short term but no improvement in the Alzheimer's Disease Assessment Scale-Cognitive Subscale behavioral test. Here, we explore a potential direct effect of Ator on Aß42 aggregation. Using NMR-based monomer consumption assays and CD spectroscopy, we observed a promoting effect of Ator in its original form (Ator-calcium) on Aß42 aggregation, as expected because of the presence of calcium ions. The effect was reversed when applying a CaCO3-based calcium ion scavenging method, which was validated by the aforementioned methods as well as thioflavin-T fluorescence assays and transmission electron microscopy. We found that the aggregation was inhibited significantly when the concentration of calcium-free Ator exceeded that of Aß by at least a factor of 2. The 1H-15N heteronuclear single quantum correlation and saturation-transfer difference NMR data suggest that calcium-free Ator exerts its effect through interaction with the 16KLVF19 binding site on the Aß peptide via its aromatic rings as well as hydroxyl and methyl groups. On the other hand, molecular dynamics simulations confirmed that the increasing concentration of Ator is necessary for the inhibition of the conformational transition of Aß from an α-helix-dominant to a ß-sheet-dominant structure.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Atorvastatina , Fragmentos de Peptídeos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Atorvastatina/farmacologia , Cálcio/metabolismo , Humanos , Camundongos , Fragmentos de Peptídeos/metabolismoRESUMO
Micrometer-sized objects are widely known to exhibit chemically driven motility in systems away from equilibrium. Experimental observation of reaction-induced motility or enhancement in diffusivity at the much shorter length scale of small molecules is, however, still a matter of debate. Here, we investigate the molecular diffusivity of reactants, catalyst, and product of a model reaction, the copper-catalyzed azide-alkyne cycloaddition click reaction, and develop new NMR diffusion approaches that allow the probing of reaction-induced diffusion enhancement in nanosized molecular systems with higher accuracy than the state of the art. Following two different approaches that enable the accounting of time-dependent concentration changes during NMR experiments, we closely monitored the diffusion coefficient of reaction components during the reaction. The reaction components showed distinct changes in the diffusivity: while the two reactants underwent a time-dependent decrease in their diffusivity, the diffusion coefficient of the product gradually increased and the catalyst showed only slight diffusion enhancement within the range expected for reaction-induced sample heating. The decrease in diffusion coefficient of the alkyne, one of the two reactants of click reaction, was not reproduced during its copper coordination when the second reactant, azide, was absent. Our results do not support the catalysis-induced diffusion enhancement of the components of the click reaction and, instead, point to the role of a relatively large intermediate species within the reaction cycle with diffusivity lower than that of both the reactants and product molecule.
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Several lines of evidence point to the important role of the N-terminal region of amyloid-beta (Aß) peptide in its toxic aggregation in Alzheimer's disease (AD). It is known that charge-altering modifications such as Ser8 phosphorylation promote Aß fibrillar aggregation. In this Letter, we combine high-pressure NMR, multiquantum chemical exchange saturation transfer (MQ-CEST) NMR, and microseconds-long molecular dynamics simulation and provide evidence of the presence of several salt bridges between Arg5 and its nearby negatively charged residues, in particular, Asp7 and Glu3. The presence of these salt bridges is correlated with less extended structures in the N-terminal region of Aß. Through density functional theory calculations, we demonstrate how the introduction of negatively charged phosphoserine 8 influences the network of adjacent salt bridges in Aß and favors more extended N-terminal structures. Our data propose a structural mechanism for the Ser8-phosphorylation-promoted Aß aggregation and define the N-terminal salt bridges as potential targets for anti-AD drug design.
Assuntos
Peptídeos beta-Amiloides/química , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Pressão , Agregados ProteicosRESUMO
The amyloid cascade hypothesis proposes that amyloid-beta (Aß) aggregation is the initial triggering event in Alzheimer's disease. Here, we utilize NMR spectroscopy and monitor the structural dynamics of two variants of Aß, Aß40 and Aß42, as a function of temperature. Despite having identical amino acid sequence except for the two additional C-terminal residues, Aß42 has higher aggregation propensity than Aß40. As revealed by the NMR data on dynamics, including backbone chemical shifts, intra-methyl cross-correlated relaxation rates and glycine-based singlet-states, the C-terminal region of Aß, especially the G33-L34-M35 segment, plays a particular role in the early steps of temperature-induced Aß aggregation. In Aß42, the distinct dynamical behaviour of C-terminal residues at higher temperatures is accompanied with marked changes in the backbone dynamics of residues V24-K28. The distinctive role of the C-terminal region of Aß42 in the initiation of aggregation defines a target for the rational design of Aß42 aggregation inhibitors.
Assuntos
Peptídeos beta-Amiloides/química , Humanos , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Dobramento de ProteínaRESUMO
Phase separation is a fundamental physicochemical process underlying the spatial arrangement and coordination of cellular events. Detailed characterization of biomolecular phase separation requires experimental access to the internal environment of dilute and especially condensed phases at high resolution. In this study, we take advantage from the ubiquitous presence of sodium ions in biomolecular samples and present the potentials of 23 Na NMR as a proxy to report the internal fluidity of biomolecular condensed phases. After establishing the temperature and viscosity dependence of 23 Na NMR relaxation rates and translational diffusion coefficient, we demonstrate that 23 Na NMR probes of rotational and translational mobility of sodium ions are capable of capturing the increasing levels of confinement in agarose gels in dependence of agarose concentration. The 23 Na NMR approach is then applied to a gel-forming phenylalanine-glycine (FG)-containing peptide, part of the nuclear pore complex involved in controlling the traffic between cytoplasm and cell nucleus. It is shown that the 23 Na NMR together with the 17 O NMR provide a detailed picture of the sodium ion and water mobility within the interior of the FG peptide hydrogel. As another example, we study phase separation in water-triethylamine (TEA) mixture and provide evidence for the presence of multiple microscopic environments within the TEA-rich phase. Our results highlight the potentials of 23 Na NMR in combination with 17 O NMR in studying biological phase separation, in particular with regards to the molecular properties of biomolecular condensates and their regulation through various physico- and biochemical factors.
Assuntos
Condensados Biomoleculares/química , Ressonância Magnética Nuclear Biomolecular , SódioRESUMO
Selectively studying parts of proteins and metabolites in tissue with nuclear magnetic resonance promises new insights into molecular structures or diagnostic approaches. Nuclear spin singlet states allow the selection of signals from chemical moieties of interest in proteins or metabolites while suppressing background signal. This selection process is based on the electron-mediated coupling between two nuclear spins and their difference in resonance frequency. We introduce a generalized and versatile pulsed NMR experiment that allows populating singlet states on a broad scale of coupling patterns. This approach allowed us to filter signals from proton pairs in the Alzheimer's disease-related b-amyloid 40 peptide and in metabolites in brain matter. In particular, for glutamine/glutamate, we have discovered a long-lived state in tissue without the typically required singlet sustaining by radiofrequency irradiation. We believe that these findings will open up new opportunities to study metabolites with a view on future in vivo applications.