1H detection and dynamic nuclear polarization-enhanced NMR of Aß1-42 fibrils.

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 ∼6.5 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.

1H-Detected Biomolecular NMR under Fast Magic-Angle Spinning.

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.

Geometry and electronic structure of Yb(III)[CH(SiMe3)2]3 from EPR and solid-state NMR augmented by computations.

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.

Structural and molecular basis of cross-seeding barriers in amyloids.

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.

Chiral Truxene-Based Self-Assembled Cages: Triple Interlocking and Supramolecular Chirogenesis.

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 (8.4 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.

A ß-barrel for oil transport through lipid membranes: Dynamic NMR structures of AlkL.

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.

Heteronuclear decoupling with rotor-synchronized phase-alternated cycles.

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.

Spectroscopic Signature and Structure of the Active Sites in Ziegler-Natta Polymerization Catalysts Revealed by Electron Paramagnetic Resonance.

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.

Monitoring Dynamics, Structure, and Magnetism of Switchable Metal-Organic Frameworks via 1 H-Detected MAS†NMR.

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 C resonances of organic linkers are detected and assigned in few hours with only 1-2 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.

Structural Analysis of an Antigen Chemically Coupled on Virus-Like Particles in Vaccine Formulation.

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.

Development of a Borane-(Meth)acrylate Photo-Click Reaction.

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.

Conformational Stability and Dynamics in Crystals Recapitulate Protein Behavior in Solution.

A growing body of evidences has established that in many cases proteins may preserve most of their function and flexibility in a crystalline environment, and several techniques are today capable to characterize molecular properties of proteins in tightly packed lattices. Intriguingly, in the case of amyloidogenic precursors, the presence of transiently populated states (hidden to conventional crystallographic studies) can be correlated to the pathological fate of the native fold; the low fold stability of the native state is a hallmark of aggregation propensity. It remains unclear, however, to which extent biophysical properties of proteins such as the presence of transient conformations or protein stability characterized in crystallo reflect the protein behavior that is more commonly studied in solution. Here, we address this question by investigating some biophysical properties of a prototypical amyloidogenic system, ß2-microglobulin in solution and in microcrystalline state. By combining NMR chemical shifts with molecular dynamics simulations, we confirmed that conformational dynamics of ß2-microglobulin native state in the crystal lattice is in keeping with what observed in solution. A comparative study of protein stability in solution and in crystallo is then carried out, monitoring the change in protein secondary structure at increasing temperature by Fourier transform infrared spectroscopy. The increased structural order of the crystalline state contributes to provide better resolved spectral components compared to those collected in solution and crucially, the crystalline samples display thermal stabilities in good agreement with the trend observed in solution. Overall, this work shows that protein stability and occurrence of pathological hidden states in crystals parallel their solution counterpart, confirming the interest of crystals as a platform for the biophysical characterization of processes such as unfolding and aggregation.

Automated Backbone NMR Resonance Assignment of Large Proteins Using Redundant Linking from a Single Simultaneous Acquisition.

Thanks to magic-angle spinning (MAS) probes with frequencies of 60-100 kHz, the benefit of high-sensitivity 1H detection can now be broadly realized in biomolecular solid-state NMR for the analysis of microcrystalline, sedimented, or lipid-embedded preparations. Nonetheless, performing the assignment of all resonances remains a rate-limiting step in protein structural studies, and even the latest optimized protocols fail to perform this step when the protein size exceeds ∼20 kDa. Here, we leverage the benefits of fast (100 kHz) MAS and high (800 MHz) magnetic fields to design an approach that lifts this limitation. Through the creation, conservation, and acquisition of independent magnetization pathways within a single triple-resonance MAS NMR experiment, a single self-consistent data set can be acquired, providing enhanced sensitivity, reduced vulnerability to machine or sample instabilities, and highly redundant linking that supports fully automated peak picking and resonance assignment. The method, dubbed RAVASSA (redundant assignment via a single simultaneous acquisition), is demonstrated with the assignment of the largest protein to date in the solid state, the 42.5 kDa maltose binding protein, using a single fully protonated microcrystalline sample and 1 week of spectrometer time.

Picometer Resolution Structure of the Coordination Sphere in the Metal-Binding Site in a Metalloprotein by NMR.

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.

Multimodal Response to Copper Binding in Superoxide Dismutase Dynamics.

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.

Paramagnetic Cobalt(II) Complexes with Cyclam Derivatives: Toward 19F MRI Contrast Agents.

In order to develop novel, more efficient, and/or selective contrast agents for magnetic resonance imaging (MRI), different modi operandi are explored as alternatives to water-relaxation enhancement. In this work, cobalt(II/III) complexes of bis(N-trifluoroethyl)cyclam derivatives with two acetate or two phosphonate pendant arms, H2te2f2a and H4te2f2p, were prepared and investigated. X-ray diffraction structures confirmed octahedral coordination with a very stable trans-III cyclam conformation and with fluorine atoms located about 5.3 Å from the metal center. The Co(II) complexes are kinetically inert, decomposing slowly even in 1 M aqueous HCl at 80 °C. The Co(II) complexes exhibited well-resolved paramagnetically shifted NMR spectra. These were interpreted with the help of quantum chemistry calculations. The 13C NMR shifts of the trans-[CoII(te2f2p)]2- complex were successfully assigned based on spin density delocalization within the ligand molecule. The obtained spin density also helps to describe d-metal-induced NMR relaxation properties of 19F nuclei, including the contribution of a Fermi contact relaxation mechanism. The paramagnetic complexes show convenient relaxation properties to be used as 19F MRI contrast agents.

Protein NMR Resonance Assignment without Spectral Analysis: 5D SOlid-State Automated Projection SpectroscopY (SO-APSY).

Narrow proton signals, high sensitivity, and efficient coherence transfers provided by fast magic-angle spinning at high magnetic fields make automated projection spectroscopy feasible for the solid-state NMR analysis of proteins. We present the first ultrahigh dimensional implementation of this approach, where 5D peak lists are reconstructed from a number of 2D projections for protein samples of different molecular sizes and aggregation states, which show limited dispersion of chemical shifts or inhomogeneous broadenings. The resulting datasets are particularly suitable to automated analysis and yield rapid and unbiased assignments of backbone resonances.

DNP NMR of biomolecular assemblies.

Dynamic Nuclear Polarization (DNP) is an effective approach to alleviate the inherently low sensitivity of solid-state NMR (ssNMR) under magic angle spinning (MAS) towards large-sized multi-domain complexes and assemblies. DNP relies on a polarization transfer at cryogenic temperatures from unpaired electrons to adjacent nuclei upon continuous microwave irradiation. This is usually made possible via the addition in the sample of a polarizing agent. The first pioneering experiments on biomolecular assemblies were reported in the early 2000s on bacteriophages and membrane proteins. Since then, DNP has experienced tremendous advances, with the development of extremely efficient polarizing agents or with the introduction of new microwaves sources, suitable for NMR experiments at very high magnetic fields (currently up to 900 MHz). After a brief introduction, several experimental aspects of DNP enhanced NMR spectroscopy applied to biomolecular assemblies are discussed. Recent demonstration experiments of the method on viral capsids, the type III and IV bacterial secretion systems, ribosome and membrane proteins are then described.

MAS dependent sensitivity of different isotopomers in selectively methyl protonated protein samples in solid state NMR.

Sensitivity and resolution together determine the quality of NMR spectra in biological solids. For high-resolution structure determination with solid-state NMR, proton-detection emerged as an attractive strategy in the last few years. Recent progress in probe technology has extended the range of available MAS frequencies up to above 100 kHz, enabling the detection of resolved resonances from sidechain protons, which are important reporters of structure. Here we characterise the interplay between MAS frequency in the newly available range of 70-110 kHz and proton content on the spectral quality obtainable on a 1 GHz spectrometer for methyl resonances. Variable degrees of proton densities are tested on microcrystalline samples of the α-spectrin SH3 domain with selectively protonated methyl isotopomers (CH3, CH2D, CHD2) in a perdeuterated matrix. The experimental results are supported by simulations that allow the prediction of the sensitivity outside this experimental frequency window. Our results facilitate the selection of the appropriate labelling scheme at a given MAS rotation frequency.

Probing Membrane Protein Insertion into Lipid Bilayers by Solid-State NMR.

Determination of the environment surrounding a protein is often key to understanding its function and can also be used to infer the structural properties of the protein. By using proton-detected solid-state NMR, we show that reduced spin diffusion within the protein under conditions of fast magic-angle spinning, high magnetic field, and sample deuteration allows the efficient measurement of site-specific exposure to mobile water and lipids. We demonstrate this site specificity on two membrane proteins, the human voltage dependent anion channel, and the alkane transporter AlkL from Pseudomonas putida. Transfer from lipids is observed selectively in the membrane spanning region, and an average lipid-protein transfer rate of 6 s-1 was determined for residues protected from exchange. Transfer within the protein, as tracked in the 15 N-1 H 2D plane, was estimated from initial rates and found to be in a similar range of about 8 to 15 s-1 for several resolved residues, explaining the site specificity.