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Protein misfolding can generate toxic intermediates, which underlies several devastating diseases, such as Alzheimer's disease (AD). The surface of AD-associated amyloid-ß peptide (Aß) fibrils has been suggested to act as a catalyzer for self-replication and generation of potentially toxic species. Specifically tailored molecular chaperones, such as the BRICHOS protein domain, were shown to bind to amyloid fibrils and break this autocatalytic cycle. Here, we identify a site on the Aß42 fibril surface, consisting of three C-terminal ß-strands and particularly the solvent-exposed ß-strand stretching from residues 26-28, which is efficiently sensed by a designed variant of Bri2 BRICHOS. Remarkably, while only a low amount of BRICHOS binds to Aß42 fibrils, fibril-catalyzed nucleation processes are effectively prevented, suggesting that the identified site acts as a catalytic aggregation hotspot, which can specifically be blocked by BRICHOS. Hence, these findings provide an understanding how toxic nucleation events can be targeted by molecular chaperones.
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Doença de Alzheimer , Amiloide , Humanos , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/genética , Domínios Proteicos , Chaperonas Moleculares/metabolismo , Fragmentos de Peptídeos/metabolismoRESUMO
Characterization of paramagnetic compounds, in particular regarding the detailed conformation and electronic structure, remains a challenge, and - still today it often relies solely on the use of X-ray crystallography, thus limiting the access to electronic structure information. This is particularly true for lanthanide elements that are often associated with peculiar structural and electronic features in relation to their partially filled f-shell. Here, we develop a methodology based on the combined use of state-of-the-art magnetic resonance spectroscopies (EPR and solid-state NMR) and computational approaches as well as magnetic susceptibility measurements to determine the electronic structure and geometry of a paramagnetic Yb(III) alkyl complex, Yb(III)[CH(SiMe3)2]3, a prototypical example, which contains notable structural features according to X-ray crystallography. Each of these techniques revealed specific information about the geometry and electronic structure of the complex. Taken together, both EPR and NMR, augmented by quantum chemical calculations, provide a detailed and complementary understanding of such paramagnetic compounds. In particular, the EPR and NMR signatures point to the presence of three-centre-two-electron Yb-γ-Me-ß-Si secondary metal-ligand interactions in this otherwise tri-coordinate metal complex, similarly to its diamagnetic Lu analogues. The electronic structure of Yb(III) can be described as a single 4f13 configuration, while an unusually large crystal-field splitting results in a thermally isolated ground Kramers doublet. Furthermore, the computational data indicate that the Yb-carbon bond contains some π-character, reminiscent of the so-called α-H agostic interaction.
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Incorporating chiral elements in host-guest systems currently attracts much attention because of the major impact such structures may have in a wide range of applications, from pharmaceuticals to materials science and beyond. Moreover, the development of multi-responsive and -functional systems is highly desirable since they offer numerous benefits. In this context, we describe herein the construction of a metal-driven self-assembled cage that associates a chiral truxene-based ligand and a bis-ruthenium complex. The maximum separation between both facing chiral units in the assembly is fixed by the intermetallic distance within the lateral bis-ruthenium complex (8.4â Å). The resulting chiral cavity was shown to encapsulate polyaromatic guest molecules, but also to afford a chiral triply interlocked [2]catenane structure. The formation of the latter occurs at high concentration, while its disassembly could be achieved by the addition of a planar achiral molecule. Interestingly the planar achiral molecule exhibits induced circular dichroism signature when trapped within the chiral cavity, thus demonstrating the ability of the cage to induce supramolecular chirogenesis.
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In this work, we present a variable-temperature 23Na NMR and variable-temperature and variable-frequency electron paramagnetic resonance (EPR) analysis of the local structure of a layered P2 Na-ion battery cathode material, Na0.67[Mg0.28Mn0.72]O2 (NMMO). For the first time, we elucidate the superstructure in this material by using synchrotron X-ray diffraction and total neutron scattering and show that this superstructure is consistent with NMR and EPR spectra. To complement our experimental data, we carry out ab initio calculations of the quadrupolar and hyperfine 23Na NMR shifts, the Na+ ion hopping energy barriers, and the EPR g-tensors. We also describe an in-house simulation script for modeling the effects of ionic mobility on variable-temperature NMR spectra and use our simulations to interpret the experimental spectra, available upon request. We find long-zigzag-type Na ordering with two different types of Na sites, one with high mobility and the other with low mobility, and reconcile the tendency toward Na+/vacancy ordering to the preservation of local electroneutrality. The combined magnetic resonance methodology for studying local paramagnetic environments from the perspective of electron and nuclear spins will be useful for examining the local structures of materials for devices.
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A new heteronuclear decoupling pulse sequence is introduced, dubbed ROtor-Synchronized Phase-Alternated Cycles (ROSPAC). It is based on a partial refocusing of the coherences (spin operator products or cross-terms) [Filip et al., J. Mag. Reson. 176, 2 (2005)] responsible for transverse spin-polarization dephasing, on the irradiation of a large pattern of radio-frequencies, and on a significant minimization of the cross-effects implying 1H chemical-shift anisotropy. Decoupling efficiency is analyzed by numerical simulations and experiments and compared to that of established decoupling sequences [swept-frequency two-pulse phase-modulated (TPPM), TPPM, small phase incremental alternation (SPINAL), refocused Continuous-wave (CWApa), and Rotor-Synchronized Hahn-Echo pulse train (RS-HEPT)]. It was found that ROSPAC offers good 1H offset robustness for a large range of chemical shifts and low radio-frequency (RF) powers, and performs very well in the ultra-fast magic-angle spinning (MAS) regime, where it is almost independent from RF power and permits it to avoid rotary-resonance recoupling conditions (v1 = nvr, n = 1, 2). It has the advantage that only the pulse lengths require optimization and has a low duty cycle in the pulsed decoupling regime. The efficiency of the decoupling sequence is demonstrated on a model microcrystalline sample of the model protein domain GB1 at 100 kHz MAS at 18.8 T.
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Ondas de Rádio , AnisotropiaRESUMO
Membrane proteins (MPs) comprise about one-third of the human proteome, playing critical roles in many physiological processes and associated disorders. Consistently, they represent one of the largest classes of targets for the pharmaceutical industry. Their study at the molecular level is however particularly challenging, resulting in a severe lack of structural and dynamic information that is hindering their detailed functional characterization and the identification of novel potent drug candidates.Magic Angle Spinning (MAS) NMR is a reliable and efficient method for the determination of protein structures and dynamics and for the identification of ligand binding sites and equilibria. MAS-NMR is particularly well suited for MPs since they can be directly analysed in a native-like lipid bilayer environment but used to require aggravating large amounts of isotope enriched material. The frequent toxicity of human MP overexpression in bacterial cultures poses an additional hurdle, resulting in the need for alternative (and often more costly) expression systems. The recent development of very fast (up to 150 kHz) MAS probes has revolutionized the field of biomolecular solid-state NMR enabling higher spectral resolution with significant reduction of the required sample, rendering eukaryotic expression systems cost-effective.Here is presented a set of accessible procedures validated for the production and preparation of eukaryotic MPs for Fast-MAS 1H-detected NMR analysis. The methodology is illustrated with the human copper uptake protein hCTR1 recombinantly produced and 13C-15N uniformly labeled with the versatile and affordable Pichia pastoris system. Subsequent purification procedures allow the recovery of mg amounts that are then reconstituted into liposome formulations compatible with solid-state NMR handling and analysis.
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Proteínas de Membrana , Saccharomycetales , Humanos , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Pichia/metabolismoRESUMO
Since the first pioneering studies on small deuterated peptides dating more than 20 years ago, 1H detection has evolved into the most efficient approach for investigation of biomolecular structure, dynamics, and interactions by solid-state NMR. The development of faster and faster magic-angle spinning (MAS) rates (up to 150 kHz today) at ultrahigh magnetic fields has triggered a real revolution in the field. This new spinning regime reduces the 1H-1H dipolar couplings, so that a direct detection of 1H signals, for long impossible without proton dilution, has become possible at high resolution. The switch from the traditional MAS NMR approaches with 13C and 15N detection to 1H boosts the signal by more than an order of magnitude, accelerating the site-specific analysis and opening the way to more complex immobilized biological systems of higher molecular weight and available in limited amounts. This paper reviews the concepts underlying this recent leap forward in sensitivity and resolution, presents a detailed description of the experimental aspects of acquisition of multidimensional correlation spectra with fast MAS, and summarizes the most successful strategies for the assignment of the resonances and for the elucidation of protein structure and conformational dynamics. It finally outlines the many examples where 1H-detected MAS NMR has contributed to the detailed characterization of a variety of crystalline and noncrystalline biomolecular targets involved in biological processes ranging from catalysis through drug binding, viral infectivity, amyloid fibril formation, to transport across lipid membranes.
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Proteínas , Prótons , Espectroscopia de Ressonância Magnética , Ressonância Magnética Nuclear Biomolecular/métodos , Peptídeos , Proteínas/químicaRESUMO
The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg2+ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). We find that neither the Mg2+-bound closed structure nor the Mg2+-free open forms are sufficient to explain the average conformation of CorA. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg2+. We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg2+ but that conducting states become more populated in Mg2+-free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.
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Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Magnésio/metabolismo , Transporte Biológico , Espectroscopia de Ressonância Magnética , Modelos Químicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
Several publications describing high-resolution structures of amyloid-ß (Aß) and other fibrils have demonstrated that magic-angle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments-namely, 1H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive two-dimensional (2D) and three-dimensional (3D) correlations obtained on 13C,15N-enriched, and fully protonated samples of M0Aß1-42 fibrils by high-field 1H-detected NMR at 23.4 T and 18.8 T, and 13C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of M0Aß1-42 (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently â¼6.5 times more sensitive than 1H detection. These results suggest that 1H detection and DNP may be the spectroscopic approaches of choice for future studies of Aß and other amyloid systems.
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Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Espectroscopia de Prótons por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13/métodos , Conformação Proteica , TemperaturaRESUMO
We present a toolbox for the rapid characterisation of powdered samples of paramagnetic metal-organic frameworks at natural abundance by 1 H-detected solid-state NMR. Very fast MAS rates at room and cryogenic temperatures and a set of tailored radiofrequency irradiation schemes help overcome the sensitivity and resolution limits often associated with the characterisation of MOF materials. We demonstrate the approach on DUT-8(Ni), a framework containing Ni2+ paddle-wheel units which can exist in two markedly different architectures. Resolved 1 H and 13 Câ resonances of organic linkers are detected and assigned in few hours with only 1-2â mg of sample at natural isotopic abundance, and used to rapidly extract information on structure and local internal dynamics of the assemblies, as well as to elucidate the metal electronic properties over an extended temperature range. The experiments disclose new possibilities for describing local and global structural changes and correlating them to electronic and magnetic properties of the assemblies.
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Despite decades of extensive studies, the atomic-scale structure of the active sites in heterogeneous Ziegler-Natta (ZN) catalysts, one of the most important processes of the chemical industry, remains elusive and a matter of debate. In the present work, the structure of active sites of ZN catalysts in the absence of ethylene, referred to as dormant active sites, is elucidated from magnetic resonance experiments carried out on samples reacted with increasing amounts of BCl3 so as to enhance the concentration of active sites and observe clear spectroscopic signatures. Using electron paramagnetic resonance (EPR) and NMR spectroscopies, in particular 2D HYSCORE experiments complemented by density functional theory (DFT) calculations, we show that the activated ZN catalysts contain bimetallic alkyl-Ti(III),Al species whose amount is directly linked to the polymerization activity of MgCl2-supported Ziegler-Natta catalysts. This connects those spectroscopic signatures to the active species formed in the presence of ethylene and enables us to propose an ethylene polymerization mechanism on the observed bimetallic alkyl-Ti(III),Al species based on DFT computations.
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In the development of 3D printing fuels, there is a need for new photoinitiating systems working under mild conditions and/or leading to polymers with new and/or enhanced properties. In this context, we introduce herein N-heterocyclic carbene-borane complexes as reagents for a new type of photo-click reaction, the borane-(meth)acrylate click reaction. Remarkably, the higher bond number of boranes relative to thiols induced an increase of the network density associated with faster polymerization kinetics. Solid-state NMR evidenced the strong participation of the boron centers on the network properties, while DMA and AFM showed that the materials exhibit improved mechanical properties, as well as reduced solvent swelling.
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Magic-Angle Spinning (MAS) Nuclear Magnetic Resonance (NMR) is a fast-developing technique, capable of complementing solution NMR, X-ray crystallography, and electron microscopy for the biophysical characterization of microcrystalline, poorly crystalline or disordered protein samples, such as enzymes, biomolecular assemblies, membrane-embedded systems or fibrils. Beyond structures, MAS NMR is an ideal tool for the investigation of dynamics, since it is unique in its ability to distinguish static and dynamic disorder, and to characterize not only amplitudes but also timescales of motion. Building on seminal work on model proteins, the technique is now ripe for widespread application in structural biology. This review briefly summarizes the recent evolutions in biomolecular MAS NMR and accounts for the growing number of systems where this spectroscopy has provided a description of conformational dynamics over the very last few years.
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Proteínas , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Movimento (Física) , Ressonância Magnética Nuclear BiomolecularRESUMO
The E.coli maltose binding protein (MBP) is a 42.5 kDa molecule widely employed in many biotechnology applications. Because of its molecular size, it has become the main model system for the development of solution NMR methods adapted to large biomolecular targets. Here, we report virtually complete (~ 90%) backbone resonance assignments obtained on a microcrystalline sample of MBP with 1H-detected solid-state NMR at fast (> 100 kHz) magic-angle spinning. We additionally present the detailed description of the methodology employed for the preparation of the sample and the acquisition and analysis of the NMR spectra. The chemical shifts, obtained with a single uniformly 15N, 13C-labelled and fully-protonated sample and about 2 weeks on a 800 MHz NMR spectrometer, have been deposited to the BMRB under the accession number 50089.
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Proteínas Ligantes de MaltoseRESUMO
Structure determination of adjuvant-coupled antigens is essential for rational vaccine development but has so far been hampered by the relatively low antigen content in vaccine formulations and by their heterogeneous composition. Here we show that magic-angle spinning (MAS) solid-state NMR can be used to assess the structure of the influenza virus hemagglutinin stalk long alpha helix antigen, both in its free, unformulated form and once chemically coupled to the surface of large virus-like particles (VLPs). The sensitivity boost provided by high-field dynamic nuclear polarization (DNP) and proton detection at fast MAS rates allows to overcome the penalty associated with the antigen dilution. Comparison of the MAS NMR fingerprints between the free and VLP-coupled forms of the antigen provides structural evidence of the conservation of its native fold upon bioconjugation. This work demonstrates that high-sensitivity MAS NMR is ripe to play a major role in vaccine design, formulation studies, and manufacturing process development.
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Antígenos Virais/análise , Vacinas de Partículas Semelhantes a Vírus/química , Ressonância Magnética Nuclear BiomolecularRESUMO
Neurodegenerative disorders are frequently associated with ß-sheet-rich amyloid deposits. Amyloid-forming proteins can aggregate under different structural conformations known as strains, which can exhibit a prion-like behavior and distinct pathophenotypes. Precise molecular determinants defining strain specificity and cross-strain interactions (cross-seeding) are currently unknown. The HET-s prion protein from the fungus Podospora anserina represents a model system to study the fundamental properties of prion amyloids. Here, we report the amyloid prion structure of HELLF, a distant homolog of the model prion HET-s. We find that these two amyloids, sharing only 17% sequence identity, have nearly identical ß-solenoid folds but lack cross-seeding ability in vivo, indicating that prion specificity can differ in extremely similar amyloid folds. We engineer the HELLF sequence to explore the limits of the sequence-to-fold conservation and to pinpoint determinants of cross-seeding and prion specificity. We find that amyloid fold conservation occurs even at an exceedingly low level of identity to HET-s (5%). Next, we derive a HELLF-based sequence, termed HEC, able to breach the cross-seeding barrier in vivo between HELLF and HET-s, unveiling determinants controlling cross-seeding at residue level. These findings show that virtually identical amyloid backbone structures might not be sufficient for cross-seeding and that critical side-chain positions could determine the seeding specificity of an amyloid fold. Our work redefines the conceptual boundaries of prion strain and sheds light on key molecular features concerning an important class of pathogenic agents.
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Amiloide/química , Amiloide/metabolismo , Príons/metabolismo , Sequência de Aminoácidos/genética , Amiloide/ultraestrutura , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/metabolismo , Sequência Conservada/genética , Proteínas Fúngicas/metabolismo , Modelos Biológicos , Podospora/genética , Agregados Proteicos/fisiologia , Dobramento de Proteína , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Fast (60 kHz) magic angle spinning solid-state NMR allows very sensitive proton detection in highly paramagnetic organometallic powders. We showcase this technique with the complete assignment of 1H and 13C resonances in a high-spin Fe(ii) polymerisation catalyst with less than 2 mg of sample at natural abundance.
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Copper/zinc superoxide dismutase (SOD) is a homodimeric metalloenzyme that has been extensively studied as a benchmark for structure-function relationships in proteins, in particular because of its implication in the familial form of the neurodegenerative disease amyotrophic lateral sclerosis. Here, we investigate microcrystalline preparations of two differently metalated forms of SOD, namely, the fully mature functional Cu,Zn state and the E,Zn-SOD state in which the Cu site is empty. By using solid-state NMR with fast magic-angle spinning (MAS) at high magnetic fields (1H Larmor frequency of 800-1000 MHz), we quantify motions spanning a dynamic range from ns to ms. We determine that metal ion uptake does not act as a rigidification element but as a switch redistributing motional processes on different time scales, with coupling of the dynamics of histidine side chains and those of remote key backbone elements of the protein.
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Esclerose Lateral Amiotrófica/metabolismo , Cobre/química , Histidina/química , Superóxido Dismutase/química , Zinco/química , Sítios de Ligação , Cristalização , Humanos , Cinética , Campos Magnéticos , Espectroscopia de Ressonância Magnética , Metaloproteínas/química , Modelos Moleculares , Conformação Proteica , Multimerização ProteicaRESUMO
Most of our understanding of chemistry derives from atomic-level structures obtained with single-crystal X-ray diffraction. Metal centers in X-ray structures of small organometallic or coordination complexes are often extremely well-defined, with errors in the positions on the order of 10-4-10-5 Å. Determining the metal coordination geometry to high accuracy is essential for understanding metal center reactivity, as even small structural changes can dramatically alter the metal activity. In contrast, the resolution of X-ray structures in proteins is limited typically to the order of 10-1 Å. This resolution is often not sufficient to develop precise structure-activity relations for the metal sites in proteins, because the uncertainty in positions can cover all of the known ranges of bond lengths and bond angles for a given type of metal complex. Here we introduce a new approach that enables the determination of a high-definition structure of the active site of a metalloprotein from a powder sample, by combining magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, tailored radio frequency (RF) irradiation schemes, and computational approaches. This allows us to overcome the "blind sphere" in paramagnetic proteins, and to observe and assign 1H, 13C, and 15N resonances for the ligands directly coordinating the metal center. We illustrate the method by determining the bond lengths in the structure of the CoII coordination sphere at the core of human superoxide dismutase 1 (SOD) with 0.7 pm precision. The coordination geometry of the resulting structure explains the nonreactive nature of the CoII/ZnII centers in these proteins, which allows them to play a purely structural role.
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Cobalto/química , Complexos de Coordenação/química , Metaloproteínas/química , Superóxido Dismutase-1/química , Zinco/química , Sítios de Ligação , Humanos , Ressonância Magnética Nuclear BiomolecularRESUMO
The protein AlkL is known to increase permeability of the outer membrane of bacteria for hydrophobic molecules, yet the mechanism of transport has not been determined. Differing crystal and NMR structures of homologous proteins resulted in a controversy regarding the degree of structure and the role of long extracellular loops. Here we solve this controversy by determining the de novo NMR structure in near-native lipid bilayers, and by accessing structural dynamics relevant to hydrophobic substrate permeation through molecular-dynamics simulations and by characteristic NMR relaxation parameters. Dynamic lateral exit sites large enough to accommodate substrates such as carvone or octane occur through restructuring of a barrel extension formed by the extracellular loops.