Ultrafast Magic Angle Spinning Solid-State NMR Spectroscopy: Advances in Methodology and Applications.

Solid-state NMR spectroscopy is one of the most commonly used techniques to study the atomic-resolution structure and dynamics of various chemical, biological, material, and pharmaceutical systems spanning multiple forms, including crystalline, liquid crystalline, fibrous, and amorphous states. Despite the unique advantages of solid-state NMR spectroscopy, its poor spectral resolution and sensitivity have severely limited the scope of this technique. Fortunately, the recent developments in probe technology that mechanically rotate the sample fast (100 kHz and above) to obtain "solution-like" NMR spectra of solids with higher resolution and sensitivity have opened numerous avenues for the development of novel NMR techniques and their applications to study a plethora of solids including globular and membrane-associated proteins, self-assembled protein aggregates such as amyloid fibers, RNA, viral assemblies, polymorphic pharmaceuticals, metal-organic framework, bone materials, and inorganic materials. While the ultrafast-MAS continues to be developed, the minute sample quantity and radio frequency requirements, shorter recycle delays enabling fast data acquisition, the feasibility of employing proton detection, enhancement in proton spectral resolution and polarization transfer efficiency, and high sensitivity per unit sample are some of the remarkable benefits of the ultrafast-MAS technology as demonstrated by the reported studies in the literature. Although the very low sample volume and very high RF power could be limitations for some of the systems, the advantages have spurred solid-state NMR investigation into increasingly complex biological and material systems. As ultrafast-MAS NMR techniques are increasingly used in multidisciplinary research areas, further development of instrumentation, probes, and advanced methods are pursued in parallel to overcome the limitations and challenges for widespread applications. This review article is focused on providing timely comprehensive coverage of the major developments on instrumentation, theory, techniques, applications, limitations, and future scope of ultrafast-MAS technology.

Solid-State NMR: Methods for Biological Solids.

In the last two decades, solid-state nuclear magnetic resonance (ssNMR) spectroscopy has transformed from a spectroscopic technique investigating small molecules and industrial polymers to a potent tool decrypting structure and underlying dynamics of complex biological systems, such as membrane proteins, fibrils, and assemblies, in near-physiological environments and temperatures. This transformation can be ascribed to improvements in hardware design, sample preparation, pulsed methods, isotope labeling strategies, resolution, and sensitivity. The fundamental engagement between nuclear spins and radio-frequency pulses in the presence of a strong static magnetic field is identical between solution and ssNMR, but the experimental procedures vastly differ because of the absence of molecular tumbling in solids. This review discusses routinely employed state-of-the-art static and MAS pulsed NMR methods relevant for biological samples with rotational correlation times exceeding 100's of nanoseconds. Recent developments in signal filtering approaches, proton methodologies, and multiple acquisition techniques to boost sensitivity and speed up data acquisition at fast MAS are also discussed. Several examples of protein structures (globular, membrane, fibrils, and assemblies) solved with ssNMR spectroscopy have been considered. We also discuss integrated approaches to structurally characterize challenging biological systems and some newly emanating subdisciplines in ssNMR spectroscopy.

Separating an overlapped 1H peak and identifying its 1H-1H correlations with the use of single-channel 1H solid-state NMR at fast MAS.

Fast magic-angle spinning (≥60 â€‹kHz) technique has enabled the acquisition of high-resolution 1H NMR spectra of solid materials. However, the spectral interpretation is still difficult because the 1H peaks are overlapped due to the narrow chemical shift range and broad linewidths. An additional 13C or 14N or 1H dimension possibly addresses the issues of overlapped proton resonances, but it leads to the elongated experimental time. Herein, we introduce a single-channel 1H experiment to separate the overlapped 1H peak and identify its spatially proximal 1H-1H correlations. This sequence combines selective excitation, selective 1H-1H polarization transfer by selective recoupling of protons (SERP), and broadband 1H recoupling by back-to-back (BABA) recoupling sequences. The concept for 1H separation is based on (i) the selective excitation of a well-resolved 1H peak and (ii) the selective dipolar polarization transfer from this isolated 1H peak to one of the 1H peaks in the overlapped/poor resolution region by SERP and (iii) the detection of 1H-1H correlations from these two 1H peaks to other neighboring 1Hs by BABA. We demonstrated the applicability of this approach to identify overlapped peaks on two molecules, ß-L-aspartyl-l-alanine and Pioglitazone.HCl. The sequence allows the clear observation of 1H-1H correlations from an overlapped 1H peak without an additional heteronuclear dimension and ensures efficient polarization transfers that leads to twelve fold reduction in experimental time compared to 14N edited experiments. The limitation and the conditions of applicability for this approach are discussed in detail.

Efficient symmetry-based γ-encoded DQ recoupling sequences for suppression of t1-noise in solid-state NMR spectroscopy at fast MAS.

Solid-state NMR spectroscopy has played a significant role in elucidating the structure and dynamics of materials and biological solids at a molecular level for decades. In particular, the 1H double-quantum/single-quantum (DQ/SQ) chemical shift correlation experiment is widely used for probing the proximity of protons, rendering it a powerful tool for elucidating the hydrogen-bonding interactions and molecular packing of various complex molecular systems. Two factors, namely, the DQ filtering efficiency and t1-noise, dictate the quality of the 2D 1H DQ/SQ spectra. Experimentally different recoupling sequences show varied DQ filtering efficiencies and t1-noise. Herein, after a systematic search of symmetry-based DQ recoupling sequences, we report that the symmetry-based γ-encoded RNnν sequences show superior performance to other DQ recoupling sequences, which not only have a higher DQ recoupling efficiency but can also significantly reduce t1-noise. The origin of t1-noise is further discussed in detail via extensive numerical simulations. We envisage that such γ-encoded RNnν sequences are superior candidates for DQ recoupling in proton-based solid-state NMR spectroscopy due to its capability of efficiently exciting DQ coherences and suppressing t1-noise.

Data driven forecasting of aperiodic motions of non-autonomous systems.

In the present effort, a data-driven modeling approach is undertaken to forecast aperiodic responses of non-autonomous systems. As a representative non-autonomous system, a harmonically forced Duffing oscillator is considered. Along with it, an experimental prototype of a Duffing oscillator is studied. Data corresponding to chaotic motions are obtained through simulations of forced oscillators with hardening and softening characteristics and experiments with a bistable oscillator. Portions of these datasets are used to train a neural machine and make response predictions and forecasts for motions on the corresponding attractors. The neural machine is constructed by using a deep recurrent neural network architecture. The experiments conducted with the different numerical and experimental chaotic time-series data confirm the effectiveness of the constructed neural network for the forecasting of non-autonomous system responses.

Simultaneous recording of intra- and inter-residue linking experiments for backbone assignments in proteins at MAS frequencies higher than 60 kHz.

Obtaining site-specific assignments for the NMR spectra of proteins in the solid state is a significant bottleneck in deciphering their biophysics. This is primarily due to the time-intensive nature of the experiments. Additionally, the low resolution in the [Formula: see text]-dimension requires multiple complementary experiments to be recorded to lift degeneracies in assignments. We present here an approach, gleaned from the techniques used in multiple-acquisition experiments, which allows the recording of forward and backward residue-linking experiments in a single experimental block. Spectra from six additional pathways are also recovered from the same experimental block, without increasing the probe duty cycle. These experiments give intra- and inter residue connectivities for the backbone [Formula: see text], [Formula: see text], [Formula: see text] and [Formula: see text] resonances and should alone be sufficient to assign these nuclei in proteins at MAS frequencies > 60 kHz. The validity of this approach is tested with experiments on a standard tripeptide N-formyl methionyl-leucine-phenylalanine (f-MLF) at a MAS frequency of 62.5 kHz, which is also used as a test-case for determining the sensitivity of each of the experiments. We expect this approach to have an immediate impact on the way assignments are obtained at MAS frequencies [Formula: see text].

Accuracy of 1H-1H distances measured using frequency selective recoupling and fast magic-angle spinning.

Selective recoupling of protons (SERP) is a method to selectively and quantitatively measure magnetic dipole-dipole interaction between protons and, in turn, the proton-proton distance in solid-state samples at fast magic-angle spinning. We present a bimodal operator-based Floquet approach to describe the numerically optimized SERP recoupling sequence. The description calculates the allowed terms in the first-order effective Hamiltonian, explains the origin of selectivity during recoupling, and shows how different terms are modulated as a function of the radio frequency amplitude and the phase of the sequence. Analytical and numerical simulations have been used to evaluate the effect of higher-order terms and offsets on the polarization transfer efficiency and quantitative distance measurement. The experimentally measured 1H-1H distances on a fully protonated thymol sample are ∼10%-15% shorter than those reported from diffraction studies. A semi-quantitative model combined with extensive numerical simulations is used to rationalize the effect of the third-spin and the role of different parameters in the experimentally observed shorter distances. Measurements at high magnetic fields improve the match between experimental and diffraction distances. The measurement of 1H-1H couplings at offsets different from the SERP-offset has also been explored. Experiments were also performed on a perdeuterated ubiquitin sample to demonstrate the feasibility of simultaneously measuring multiple quantitative distances and to evaluate the accuracy of the measured distance in the absence of multispin effects. The estimation of proton-proton distances provides a boost to structural characterization of small pharmaceuticals and biomolecules, given that the positions of protons are generally not well defined in x-ray structures.

Measuring strong one-bond dipolar couplings using REDOR in magic-angle spinning solid-state NMR.

Rotational-Echo DOuble Resonance, REDOR, is an experimentally robust and a well-established dipolar-recoupling technique to measure dipolar couplings between isolated pairs of spin-1/2 heteronuclei in solid-state nuclear magnetic resonance. REDOR can also be used to estimate motional order parameters when the bond distance is known, for example, in the case of directly bound nuclei. However, the relatively fast dipolar dephasing for strongly coupled spin-1/2 pairs, such as 13C-1H, makes the stroboscopic measurement required in this experiment challenging, even at fast Magic-Angle-Spinning (MAS) frequencies. In such cases, modified REDOR-based methods like Shifted-REDOR (S-REDOR) are used to scale the dipolar coupling compared to REDOR. This is achieved by changing the position of one of the two recoupling π-pulses in a rotor period. This feature, however, comes at the cost of mixing multiple Fourier components of the dipolar coupling and can, additionally, require high radio-frequency amplitudes to realise small scaling factors. We introduce here a general pulse scheme which involves shifting both the π pulses in the REDOR scheme to achieve arbitrary scaling factors whilst retaining the robustness and simplicity of REDOR recoupling and avoiding the disadvantages of S-REDOR. The classical REDOR is a specific case of this scheme with a scaling factor of one. We demonstrate the results on isolated 13C-15N and 1H-13C spin pairs at 20 and 62.5 kHz MAS, respectively.

Efficient low-power TOBSY sequences for fast MAS.

Through-bond J-coupling based experiments in solid-state NMR spectroscopy are challenging because the J couplings are typically much smaller than the dipolar couplings. This often leads to a lower transfer efficiency compared to dipolar-coupling based sequences. One of the reasons for the low transfer efficiency are the second-order cross terms involving the strong heteronuclear dipolar couplings leading to fast magnetization decay. Here, we show that by employing a symmetry-based C9 sequence, which was carefully selected to suppress second-order terms, efficient polarization transfers of up to 80% can be achieved without decoupling on fully protonated two-spin model systems at a MAS frequency of 55.5 kHz with rf-field amplitudes of about 25 kHz. In addition, we analyse the effects of rf inhomogeneity and crystallites selection due to the polarization preparation method on the TOBSY transfer efficiency. We demonstrate on small model substances as well as on deuterated and 100% back-exchanged ubiquitin that C9391 and C9481 are efficient and practical TOBSY sequences at experimental conditions ranging from proton Larmor frequencies of 400-850 MHz, and MAS frequencies ranging from 55.5 to 111.1 kHz.

Cytotoxic Oligomers and Fibrils Trapped in a Gel-like State of α-Synuclein Assemblies.

α-Synuclein (α-Syn) aggregation is associated with Parkinson's disease (PD) pathogenesis. In PD, the role of oligomers versus fibrils in neuronal cell death is debatable, but recent studies suggest oligomers are a proximate neurotoxin. Herein, we show that soluble α-Syn monomers undergo a transformation from a solution to a gel state on incubation at high concentration. Detailed characterization of the gel showed the coexistence of monomers, oligomers, and short fibrils. In vitro, the gel was highly cytotoxic to human neuroblastoma cells. The individual constituents of the gel are short-lived species but toxic to the cells. They comprise a structurally heterogeneous population of α-helical and ß-sheet-rich oligomers and short fibrils with the cross-ß motif. Given the recent evidence of the gel-like state of the protein associated with neurodegenerative diseases, the gel state of α-Syn in this study represents a mechanistic and structural model for the in vivo toxicity of α-Syn in PD.

Parameter independent low-power heteronuclear decoupling for fast magic-angle spinning solid-state NMR.

Major advances have recently been made in the field of heteronuclear dipolar decoupling in solid-state nuclear magnetic resonance (NMR). These developments have improved the resolution and sensitivity of the NMR spectrum of spins coupled to protons. One such new scheme, denoted as rCWApA, has proven to be robust with practically no need for parameter optimization [A. Equbal et al. Chem. Phys. Lett., 635, 339 (2015)]. Most of the experiments with rCWApA have been carried out in the regimes of slow to moderate magic-angle spinning while simultaneously applying high decoupling radio-frequency amplitudes. Here, we explore the performance of the rCWApA sequence and its predecessor rCWA in the regime of low-power radio-frequency irradiation and fast magic-angle spinning. The robustness of the refocused continuous-wave (rCW) schemes to experimental parameters such as pulse lengths and offset irradiation is demonstrated. Numerical simulations and analytical theory have been used to understand the effects of various nuclear spin interactions on the decoupling performance of the low-power rCW decoupling scheme relative to other decoupling methods. This has lead to the design of an "optimum low-power decoupling sequence" that can be used without parameter optimization. This result is particularly important in the context of samples with low signal to noise.

Sine-squared shifted pulses for recoupling interactions in solid-state NMR.

Rotational-Echo DOuble-Resonance (REDOR) is a versatile experiment for measuring internuclear distance between two heteronuclear spins in solid-state NMR. At slow to intermediate magic-angle spinning (MAS) frequencies, the measurement of distances between strongly coupled spins is challenging due to rapid dephasing of magnetisation. This problem can be remedied by employing the pulse-shifted version of REDOR known as Shifted-REDOR (S-REDOR) that scales down the recoupled dipolar coupling. In this study, we propose a new variant of the REDOR sequence where the positions of the π pulses are determined by a sine-squared function. This new variant has scaling properties similar to S-REDOR. We use theory, numerical simulations, and experiments to compare the dipolar recoupling efficiencies and the experimental robustness of the three REDOR schemes. The proposed variant has advantages in terms of radiofrequency field requirements at fast MAS frequencies.

Accelerating proton spin diffusion in perdeuterated proteins at 100 kHz MAS.

Fast magic-angle spinning (>60 kHz) has many advantages but makes spin-diffusion-type proton-proton long-range polarization transfer inefficient and highly dependent on chemical-shift offset. Using 100%-HN-[2H,13C,15N]-ubiquitin as a model substance, we quantify the influence of the chemical-shift difference on the spin diffusion between proton spins and compare two experiments which lead to an improved chemical-shift compensation of the transfer: rotating-frame spin diffusion and a new experiment, reverse amplitude-modulated MIRROR. Both approaches enable broadband spin diffusion, but the application of the first variant is limited due to fast spin relaxation in the rotating frame. The reverse MIRROR experiment, in contrast, is a promising candidate for the determination of structurally relevant distance restraints. The applied tailored rf-irradiation schemes allow full control over the range of recoupled chemical shifts and efficiently drive spin diffusion. Here, the relevant relaxation time is the larger longitudinal relaxation time, which leads to a higher signal-to-noise ratio in the spectra.

A generalized theoretical framework for the description of spin decoupling in solid-state MAS NMR: Offset effect on decoupling performance.

We present a generalized theoretical framework that allows the approximate but rapid analysis of residual couplings of arbitrary decoupling sequences in solid-state NMR under magic-angle spinning conditions. It is a generalization of the tri-modal Floquet analysis of TPPM decoupling [Scholz et al., J. Chem. Phys. 130, 114510 (2009)] where three characteristic frequencies are used to describe the pulse sequence. Such an approach can be used to describe arbitrary periodic decoupling sequences that differ only in the magnitude of the Fourier coefficients of the interaction-frame transformation. It allows a ∼100 times faster calculation of second-order residual couplings as a function of pulse sequence parameters than full spin-dynamics simulations. By comparing the theoretical calculations with full numerical simulations, we show the potential of the new approach to examine the performance of decoupling sequences. We exemplify the usefulness of this framework by analyzing the performance of commonly used high-power decoupling sequences and low-power decoupling sequences such as amplitude-modulated XiX (AM-XiX) and its super-cycled variant SC-AM-XiX. In addition, the effect of chemical-shift offset is examined for both high- and low-power decoupling sequences. The results show that the cross-terms between the dipolar couplings are the main contributions to the line broadening when offset is present. We also show that the SC-AM-XIX shows a better offset compensation.

Protein resonance assignment at MAS frequencies approaching 100 kHz: a quantitative comparison of J-coupling and dipolar-coupling-based transfer methods.

We discuss the optimum experimental conditions to obtain assignment spectra for solid proteins at magic-angle spinning (MAS) frequencies around 100 kHz. We present a systematic examination of the MAS dependence of the amide proton T 2' times and a site-specific comparison of T 2' at 93 kHz versus 60 kHz MAS frequency. A quantitative analysis of transfer efficiencies of building blocks, as they are used for typical 3D experiments, was performed. To do this, we compared dipolar-coupling and J-coupling based transfer steps. The building blocks were then combined into 3D experiments for sequential resonance assignment, where we evaluated signal-to-noise ratio and information content of the different 3D spectra in order to identify the best assignment strategy. Based on this comparison, six experiments were selected to optimally assign the model protein ubiquitin, solely using spectra acquired at 93 kHz MAS. Within 3 days of instrument time, the required spectra were recorded from which the backbone resonances have been assigned to over 96%.

Site-specific analysis of heteronuclear Overhauser effects in microcrystalline proteins.

Relaxation parameters such as longitudinal relaxation are susceptible to artifacts such as spin diffusion, and can be affected by paramagnetic impurities as e.g. oxygen, which make a quantitative interpretation difficult. We present here the site-specific measurement of [(1)H](13)C and [(1)H](15)N heteronuclear rates in an immobilized protein. For methyls, a strong effect is expected due to the three-fold rotation of the methyl group. Quantification of the [(1)H](13)C heteronuclear NOE in combination with (13)C-R 1 can yield a more accurate analysis of side chain motional parameters. The observation of significant [(1)H](15)N heteronuclear NOEs for certain backbone amides, as well as for specific asparagine/glutamine sidechain amides is consistent with MD simulations. The measurement of site-specific heteronuclear NOEs is enabled by the use of highly deuterated microcrystalline protein samples in which spin diffusion is reduced in comparison to protonated samples.

De†novo 3D structure determination from sub-milligram protein samples by solid-state 100†kHz MAS NMR spectroscopy.

Solid-state NMR spectroscopy is an emerging tool for structural studies of crystalline, membrane-associated, sedimented, and fibrillar proteins. A major limitation for many studies is still the large amount of sample needed for the experiments, typically several isotopically labeled samples of 10-20 mg each. Here we show that a new NMR probe, pushing magic-angle sample rotation to frequencies around 100 kHz, makes it possible to narrow the proton resonance lines sufficiently to provide the necessary sensitivity and spectral resolution for efficient and sensitive proton detection. Using restraints from such spectra, a well-defined de novo structure of the model protein ubiquitin was obtained from two samples of roughly 500 µg protein each. This proof of principle opens new avenues for structural studies of proteins available in microgram, or tens of nanomoles, quantities that are, for example, typically achieved for eukaryotic membrane proteins by in-cell or cell-free expression.

Solid-state NMR assignment of α-synuclein polymorph prepared from helical intermediate.

Synucleinopathies are neurodegenerative diseases characterized by the accumulation of α-synuclein protein aggregates in the neurons and glial cells. Both ex vivo and in vitro α-synuclein fibrils tend to show polymorphism. Polymorphism results in structure variations among fibrils originating from a single polypeptide/protein. The polymorphs usually have different biophysical, biochemical and pathogenic properties. The various pathologies of a single disease might be associated with distinct polymorphs. Similarly, in the case of different synucleinopathies, each condition might be associated with a different polymorph. Fibril formation is a nucleation-dependent process involving the formation of transient and heterogeneous intermediates from monomers. Polymorphs are believed to arise from heterogeneous oligomer populations because of distinct selection mechanisms in different conditions. To test this hypothesis, we isolated and incubated different intermediates during in vitro fibrillization of α-synuclein to form different polymorphs. Here, we report 13C and 15N chemical shifts and the secondary structure of fibrils prepared from the helical intermediate using solid-state nuclear magnetic spectroscopy.

PAIN with and without PAR: variants for third-spin assisted heteronuclear polarization transfer.

In this article, we describe third-spin assisted heteronuclear recoupling experiments, which play an increasingly important role in measuring long-range heteronuclear couplings, in particular (15)N-(13)C, in proteins. In the proton-assisted insensitive nuclei cross polarization (PAIN-CP) experiment (de Paëpe et al. in J Chem Phys 134:095101, 2011), heteronuclear polarization transfer is always accompanied by homonuclear transfer of the proton-assisted recoupling (PAR) type. We present a phase-alternating experiment that promotes heteronuclear (e.g. (15)N → (13)C) polarization transfer while simultaneously minimizing homonuclear (e.g.(13)C → (13)C) transfer (PAIN without PAR). This minimization of homonuclear polarization transfer is based on the principle of the resonant second-order transfer (RESORT) recoupling scheme where the passive proton spins are irradiated by a phase-alternating sequence and the modulation frequency is matched to an integer multiple of the spinning frequency. The similarities and differences between the PAIN-CP and this het-RESORT experiment are discussed here.

Hydrogen bonding involving side chain exchangeable groups stabilizes amyloid quarternary structure.

The amyloid ß-peptide (Aß) is the major structural component of amyloid fibrils in the plaques of brains of Alzheimer's disease patients. Numerous studies have addressed important aspects of secondary and tertiary structure of fibrils. In electron microscopic images, fibrils often bundle together. The mechanisms which drive the association of protofilaments into bundles of fibrils are not known. We show here that amino acid side chain exchangeable groups like e.g. histidines can provide useful restraints to determine the quarternary assembly of an amyloid fibril. Exchangeable protons are only observable if a side chain hydrogen bond is formed and the respective protons are protected from exchange. The method relies on deuteration of the Aß peptide. Exchangeable deuterons are substituted with protons, before fibril formation is initiated.