Magic Angle Spinning NMR Research Articles

Changes in Secondary Structure upon Pr to Pfr Transition in Cyanobacterial Phytochrome Cph1 detected by DNP NMR.

Phytochromes perceive subtle changes in the light environment and convert them into biological signals by photoconversion between the red-light absorbing (Pr) and the far-red-absorbing (Pfr) states. In the primitive bacteriophytochromes this includes refolding of a tongue-like hairpin loop close to the chromophore, one strand of an antiparallelb-sheetbeing replaced byα-helix. However,the strand sequence in thecyanobacterial phytochrome Cph1is different from that of previously investigated bacteriophytochromes and has a higherb-sheetpropensity. Weconfirm here the transition experimentally and estimate minimum helix length usingdynamic nuclear polarisation (DNP) magic angle spinning NMR. Sample conditions were optimized for protein DNP NMR studies at high field, yielding Boltzmann enhancementseBof 19 at an NMR field of 18.801 T. Selective labelling of Trp, Ile, Arg, and Val residues with13C and15N enabledfiltering for pairs of labelled amino acids by the 3D CANCOCA technique to identify signals of the motif483Ile-Val-Arg485(IVR) present in both sheet and helix. Those signals were assigned for the Pfr state of the protein. Based on the chemical shift pattern, we confirm for Cph1 the formation of a helix covering the IVR motif.

Solid-State NMR Analysis of the Dynamics of Cofactors: Comparison of Heliobacterial and Purple Bacterial Reaction Centers.

Photosynthetic reaction centers (RCs) serve as natural engines converting solar energy to chemical energy. Understanding the principles of efficient charge separation and light-induced electron transfer (ET) between the chlorophyll-type pigments might guide the synthesis for artificial photosynthetic systems. We present detailed insight into the dynamics at the atomic level using solid-state NMR techniques applied to the RCs of Heliobacillus (Hb.) mobilis (HbRCs) and the purple bacterium Rhodobacter (R.) sphaeroides (PbRCs). It is assumed that heliobacteria were among the first phototrophic organisms; therefore, their RC can be regarded as ancient. They are constructed homodimerically with perfect C2 symmetry, enabling ET over both branches of cofactors. Modern RCs of R. sphaeroides wild-type (WT) have higher redox power and are functionally highly asymmetric. The dynamics of the cofactors in both RCs has been explored using nuclear hyperpolarization, induced by the solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) effect. Based on the individual incorporation of 13C positions of the cofactors (through supplementation by 13C-δ-aminolevulinic acid), photo-CIDNP magic-angle spinning (MAS) NMR experiments provide access to the local dynamics of the cofactors along the ET path over a broad range of time scales. Theoretical analysis of the dynamic deformation of these macrocycles is also discussed in terms of function. The dynamics observed in HbRCs appears to be correlated to ET. The cofactors in PbRC are significantly less dynamic than those in the HbRC. Relevance for efficiency and redox properties are discussed.

Dipolar Recoupling in Rotating Solids.

Magic angle spinning (MAS) nuclear magnetic resonance (NMR) has evolved significantly over the past three decades and established itself as a vital tool for the structural analysis of biological macromolecules and materials. This review delves into the development and application of dipolar recoupling techniques in MAS NMR, which are crucial for obtaining detailed structural and dynamic information. We discuss a variety of homonuclear and heteronuclear recoupling methods which are essential for measuring spatial restraints and explain in detail the spin dynamics that these sequences generate. We also explore recent developments in high spinning frequency MAS, proton detection, and dynamic nuclear polarization, underscoring their importance in advancing biomolecular NMR. Our aim is to provide a comprehensive account of contemporary dipolar recoupling methods, their principles, and their application to structural biology and materials, highlighting significant contributions to the field and emerging techniques that enhance resolution and sensitivity in MAS NMR spectroscopy.

Densely packed glass structure caused by seven-coordinated Zr in high elastic modulus Al2O3–SiO2–ZrO2 glasses

Al2O3–SiO2–ZrO2 ternary glasses were fabricated using a levitation technique. The addition of ZrO2 to the Al2O3–SiO2 glasses strongly affected their thermal, mechanical, and structural properties. Several compositions were partially vitrified at the laser-melted area without levitation although their melting required temperatures higher than 2000°C. With increasing ZrO2 content, the elastic moduli linearly increased, and the 50Al2O3–20SiO2–30ZrO2 glass exhibited a Young′s modulus of 166 GPa and Vickers hardness HV of 11 GPa. Conversely, crack resistance significantly decreased with the addition of ZrO2. Density measurements, Zr L2,3-edge and K-edge X-ray absorption fine structure analyses, and 27Al and 29Si magic-angle spinning nuclear magnetic resonance spectroscopy were performed to investigate the local structure around Zr, Al, and Si in the glasses. Zr formed distorted ZrO7 as in monoclinic ZrO2, which has been rarely found in conventional oxide glasses. The highly oxygen-coordinated Al atoms such as AlO5 and AlO6, were the main components in the glasses rather than AlO4. The majority of Si atoms form SiO4 with four bridging oxygen (Q4). Among the four bridging oxygens, the number of oxygens connected to Al or Zr clearly increased with decreasing SiO2 content. The high packing density of the ternary glasses that resulted in high elastic moduli originated from the highly oxygen-coordinated Zr and Al and their close bonding with SiO4 without generating nonbridging oxygens.

Directional Transport in Hierarchically Aligned ZSM-5 Zeolites with High Catalytic Activity.

Zeolites, the most technically important crystalline microporous materials, are indispensable cornerstones of chemical engineering because of their remarkable catalytic properties and adsorption capabilities. Numerous studies have demonstrated that the hierarchical engineering of zeolites can maximize accessible active sites and improve mass transport, which significantly decreases the internal diffusion limits to achieve the desired performance. However, the construction of hierarchical zeolites with ordered alignments and size-controlled substructures in a convenient way is highly challenging. Herein, we develop a facile procedure using two common structure-directing agents, tetrapropylammonium hydroxide (TPAOH) and tetraethylammonium hydroxide (TEAOH), to synthesize hierarchically aligned ZSM-5 (Hie-ZSM-5) crystals with a-axis alignment substructures of controllable size. The control of the substructure size (α) in the range of 10-60 nm and the corresponding similarity (r = α/β, where β is the size of Hie-ZSM-5) ranging from 0.004 to 0.033 can be tuned by varying the Si/Al ratios (40-120). A systematic investigation of the overall crystallization process, using time-dependent XRD, SEM, TEM, and solid-state magic-angle spinning NMR (13C, 27Al, 29Si) methods, enable us to construct a solid mechanism for the generation of Hie-ZSM-5. Most importantly, directional transport in the unique structures of Hie-ZSM-5 efficiently enhances mass diffusion, as well as catalytic activity and stability. These findings improve our understanding of the zeolite crystallization process and inspire novel methods for the rational design of hierarchical zeolites.

Integrating 19F Distance Restraints for Accurate Protein Structure Determination by Magic Angle Spinning NMR Spectroscopy.

Traditional protein structure determination by magic angle spinning (MAS) solid-state NMR spectroscopy primarily relies on interatomic distances up to 8 Å, extracted from 13C-, 15N-, and 1H-based dipolar-based correlation experiments. Here, we show that 19F fast (60 kHz) MAS NMR spectroscopy can supply additional, longer distances. Using 4F-Trp,U-13C,15N crystalline Oscillatoria agardhii agglutinin (OAA), we demonstrate that judiciously designed 2D and 3D 19F-based dipolar correlation experiments such as (H)CF, (H)CHF, and FF can yield interatomic distances in the 8-16 Å range. Incorporation of fluorine-based restraints into structure calculation improved the precision of Trp side chain conformations as well as regions in the protein around the fluorine containing residues, with notable improvements observed for residues in proximity to the Trp pairs (W10/W17 and W77/W84) in the carbohydrate-binding loops, which lacked sufficient long-range 13C-13C distance restraints. Our work highlights the use of fluorine and 19F fast MAS NMR spectroscopy as a powerful structural biology tool.

The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19F-NMR.

Endogenous phospholipids influence the conformational equilibria of G protein-coupled receptors, regulating their ability to bind drugs and form signaling complexes. However, most studies of GPCR-lipid interactions have been carried out in mixed micelles or lipid nanodiscs. Though useful, these membrane mimetics do not fully replicate the physical properties of native cellular membranes associated with large assemblies of lipids. We investigated the conformational equilibria of the human A2A adenosine receptor (A2AAR) in phospholipid vesicles using 19F solid-state magic angle spinning NMR (SSNMR). By applying an optimized sample preparation workflow and experimental conditions, we were able to obtain 19F-SSNMR spectra for both antagonist- and agonist-bound complexes with sensitivity and linewidths closely comparable to those achieved using solution NMR. This facilitated a direct comparison of the A2AAR conformational equilibria across detergent micelle, lipid nanodisc, and lipid vesicle preparations. While antagonist-bound A2AAR showed a similar conformational equilibria across all membrane and membrane mimetic systems, the conformational equilibria of agonist-bound A2AAR exhibited differences among different environments. This suggests that the conformational equilibria of GPCRs may be influenced not only by specific receptor-lipid interactions but also by the membrane properties found in larger lipid assemblies.

Investigating the microstructure of alkali activated fly ash, ground granulated blast furnace slag (GGBS), or their mixtures by depth sensing nanoindentation and structure dependence of their composition by Magic Angle Spinning (MAS) NMR

Investigating the microstructure of alkali activated fly ash, ground granulated blast furnace slag (GGBS), or their mixtures by depth sensing nanoindentation and structure dependence of their composition by Magic Angle Spinning (MAS) NMR

Metabolomic Analysis of Histological Composition Variability of High-Grade Serous Ovarian Cancer Using 1H HR MAS NMR Spectroscopy.

In this work, the HR MAS NMR (high-resolution magic-angle spinning nuclear magnetic resonance) spectroscopy technique was combined with standard histological examinations to investigate the metabolic features of high-grade serous ovarian cancer (HGSOC) with a special focus on the relation between a metabolic profile and a cancer cell fraction. The studied group consisted of 44 patients with HGSOC and 18 patients with benign ovarian tumors. Normal ovarian tissue was also excised from 13 control patients. The metabolic profiles of 138 tissue specimens were acquired on a Bruker Avance III 400 MHz spectrometer. The NMR spectra of the HGSOC samples could be discriminated from those acquired from the non-transformed tissue and were shown to depend on tumor purity. The most important features that differentiate the samples with a high fraction of cancer cells from the samples containing mainly fibrotic stroma are the increased intensities in the spectral regions corresponding to phosphocholine/glycerophosphocholine, phosphoethanolamine/serine, threonine, uridine nucleotides and/or uridine diphosphate (UDP) nucleotide sugars. Higher levels of glutamine, glutamate, acetate, lysine, alanine, leucine and isoleucine were detected in the desmoplastic stroma within the HGSOC lesions compared to the stroma of benign tumors. The HR MAS NMR analysis of the metabolic composition of the epithelial and stromal compartments within HGSOC contributes to a better understanding of the disease's biology.

High-Efficiency Silane Utilization in Amine-Modified Adsorbents for Direct Air Capture through Interconnected Three-Dimensional Pores.

Economic synthesis of amine-modified solid adsorbents is pivotal for the global-scale direct air capture (DAC) technologies required to realize net-zero emissions. To address the problems of the traditional reflux method using excessively costly amino silane, we propose introducing silane by impregnation into mesoporous silica with interconnected three-dimensional pores. X-ray diffraction, Fourier transform infrared spectroscopy, N2 adsorption-desorption, transmission electron and scanning electron microscopies, magic-angle spinning nuclear magnetic resonance, and elemental analysis identified the spatial distribution of amino silane in the materials with different loading levels. The results of structure characterization and a comparison with a reference experiment (using a porous support with one-dimensional pores and/or the conventional reflux method) revealed that the proposed strategy provided a uniform amine distribution, together with a high utilization efficiency of the amino silane. We also demonstrate that the obtained material has a high adsorption capacity and good recycling stability comparable to those of the previously reported amino silane modified adsorbents under simulated DAC conditions.

Advanced High-Voltage Electrolyte Design Using Poly(ethylene Oxide) and High-Concentration Ionic Liquids for All-Solid-State Lithium-Metal Batteries.

Poly(ethylene oxide) (PEO)-based solid polymer electrolytes (SPEs) are among the most promising materials for solid-state lithium metal batteries (LMBs) due to their inherent safety advantages; however, they suffer from insufficient room-temperature ionic conductivity (up to 10-6 S cm-1) and limited oxidation stability (<4 V). In this study, a novel "polymer-in-high-concentrated ionic liquid (IL)" (PiHCIL) electrolyte composed of PEO, N-propyl-N-methylpyrrolidinium bis(fluorosulfonyl) imide (C3mpyrFSI) IL, and LiFSI is designed. The EO/[Li/IL] ratio has been widely varied, and physical and electrochemical properties have been explored. The Li-coordination and solvation structure has been explored through Fourier-transform infrared spectroscopy and solid-state magic-angle spinning nuclear magnetic resonance. The newly designed electrolyte provides a promisingly high oxidative stability of 5.1 V and offers high ambient temperature ionic conductivity of 5.6 × 10-4 S cm-1 at 30 °C. Li|Li symmetric cell cycling shows very stable and reversible cycling of Li metal over 100 cycles and a smooth dendrite-free deposition morphology. All-solid-state cells using a composite lithium iron phosphate cathode exhibit promising cycling with 99.2% capacity retention at a C/5 rate over 100 cycles. Therefore, the novel approach of PiHCIL enables a new pathway to design high-performing SPEs for high-energy-density all-solid-state LMBs.

Nuclear hyperpolarization in electron-transfer proteins: Revealing unexpected light-induced 15N signals with field-cycling magic-angle spinning NMR

The solid-state photo-CIDNP (photo-chemically induced dynamic nuclear polarization) effect allows for nuclear hyperpolarization, i.e., non-Boltzmann nuclear spin population. The effect relies on the light-induced formation of a spin-correlated radical pair (SCRP) and has been observed in various photosynthetic reaction center (RC) proteins and flavin-containing light, oxygen, voltage (LOV) proteins. Both systems exhibit strongly enhanced NMR signals originating from the electron transfer partners. Here, we present experimental data on the magnetic field dependence of the 15N solid-state photo-CIDNP effect in both phototropin LOV1 C57S from Chlamydomonas reinhardtii and the bacterial photosynthetic RC from Rhodobacter sphaeroides. Using a pneumatic field-cycling system, samples containing a frozen solution of the proteins are explored between 0.25 T and 9.4 T. Both systems yield hyperpolarized 15N NMR signals across the entire magnetic field range originating from the electron transfer moieties. Also, in both systems, hyperpolarized signals from unexpected positions are detected between 1.0 T and 2.0 T: position N-1 of the flavin in the LOV1 protein and the τ-N of the axial magnesium-coordinating histidine of the donor. A first attempt to explain the occurrence of these unexpected signals based on quantum chemical calculations is presented.

Expanding the tool box for native structural biology: 19F dynamic nuclear polarization with fast magic angle spinning.

Obtaining atomic-level information on components in the cell is a major focus in structural biology. Elucidating specific structural and dynamic features of proteins and their interactions in the cellular context is crucial for understanding cellular processes. We introduce 19F dynamic nuclear polarization (DNP) combined with fast magic-angle-spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy as a powerful technique to study proteins in mammalian cells. We demonstrate our approach on the severe acute respiratory syndrome coronavirus 2 5F-Trp-NNTD protein, electroporated into human cells. DNP signal enhancements of 30- to 40-fold were observed, translating into over 1000-fold experimental time savings. High signal-to-noise ratio spectra were acquired on nanomole quantities of a protein in cells in minutes. 2D 19F-19F dipolar correlation spectra with remarkable sensitivity and resolution were obtained, exhibiting 19F-19F cross peaks associated with fluorine atoms as far as ~10 angstroms apart. This work paves the way for 19F DNP-enhanced MAS NMR applications in cells for probing protein structure, dynamics, and ligand interactions.

High-Resolution Magic-Angle Spinning Nuclear Magnetic Resonance Identifies Impairment of Metabolism by T-2 Toxin, in Relation to Toxicity, in Zebrafish Embryo Model.

Among the widespread trichothecene mycotoxins, T-2 toxin is considered the most toxic congener. In the present study, we utilized high-resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR), coupled to the zebrafish (Danio rerio) embryo model, as a toxicometabolomics approach to elucidate the cellular, molecular and biochemical pathways associated with T-2 toxicity. Aligned with previous studies in the zebrafish embryo model, exposure to T-2 toxin was lethal in the high parts-per-billion (ppb) range, with a median lethal concentration (LC50) of 105 ppb. Exposure to the toxins was, furthermore, associated with system-specific alterations in the production of reactive oxygen species (ROS), including decreased ROS production in the liver and increased ROS in the brain region, in the exposed embryos. Moreover, metabolic profiling based on HRMAS NMR revealed the modulation of numerous, interrelated metabolites, specifically including those associated with (1) phase I and II detoxification, and antioxidant pathways; (2) disruption of the phosphocholine lipids of cell membranes; (3) mitochondrial energy metabolism, including apparent disruption of the tricarboxylic acid (TCA) cycle, and the electron transport chain of oxidative phosphorylation, as well as "upstream" effects on carbohydrate, i.e., glucose metabolism; and (4) several compensatory catabolic pathways. Taken together, these observations enabled development of an integrated, system-level model of T-2 toxicity in relation to human and animal health.

Effect of lithium concentration on the network connectivity of nuclear waste glasses

Structure of borosilicate glasses with varying Li2O contents were investigated using Magic Angle Spinning (MAS) Nuclear Magnetic Resonance (NMR), employing 6Li, 11B, 23Na, 27Al and 29Si nuclei. 11B MAS NMR revealed that increasing Li2O contents result in formation of [BO4]− sites at the expense of BO3. 11B{6Li} and 27Al{6Li} dipolar heteronuclear multiple quantum correlation (D-HMQC) NMR revealed Li as a charge compensator for anionic tetrahedral sites with increased Li. 11B{6Li} J-coupling mediated HMQC NMR suggested the possible association of Li with non-bridging oxygens on Q2 and Q3 sites, indicating its dual role in the glass network. 29Si and 23Na MAS NMR spectra showed depolymerisation of the silicate network and shortening of Na-O bond lengths with increased Li. NMR results are supported by Raman spectroscopy and thermal analysis that indicate depolymerisation of the silicate network and a reduction in glass transition temperature at higher Li content.

Unexpected asymmetric distribution of cholesterol and phospholipids in equilibrium model membranes

Lipid compositional asymmetry across the leaflets of the plasma membrane is a ubiquitous feature in eukaryotic cells. How this asymmetry is maintained is thought to be primarily controlled by active transport of lipids between leaflets. This strategy is facilitated by the fact that long tail phospholipids and sphingolipids diffuse through the lipid bilayer slowly – taking many hours or days. However, a lipid like cholesterol – which is the most abundant lipid in the plasma membrane of animal cells – has been harder to pin-point in terms of its favored side. In the present work we show that when a saturated lipid is added to a mix of the unsaturated lipid palmitoyl-oleoyl-phosphatidylcholine (POPC) and cholesterol, both cholesterol and the long tail phospholipids organize asymmetrically across the membrane’s leaflets naturally. In these extruded unilamellar vesicles, most cholesterol as well as the saturated lipid – dipalmitoylphosphatidylcholine (DPPC) or sphingomyelin (SM) – segregated to the inner leaflet while POPC preferentially localized in the outer leaflet. This asymmetric arrangement generated a slight phospholipid number imbalance favoring the outer leaflet and thus opposite to where cholesterol and the saturated lipids preferentially partitioned. These results were obtained using Magic Angle Spinning (MAS) NMR in combination with Small Angle Neutron Scattering (SANS) using isotope labeling to differentiate lipid species. We suggest that sidedness in membranes can be driven by thermodynamic processes. In addition, our MAS NMR results show that the lower bound for cholesterol’s flip-flop half-time at 45°C is 10ms, which is at least two orders of magnitude slower than current MD simulations predict. This result stands in stark contrast to previous work that suggested that cholesterol’s flip-flop half-time at 37°C has an upper bound of 10ms.

CO2 Dynamics in a Flexible Metal-Organic Framework with Gate-Opening Phenomenon.

As a promising porous material for CO2adsorption and storage, elastic layer-structured metal-organic framework-11 (ELM-11) has attracted significant attention owing to its distinct gate-opening phenomenon. There is a sharp increase in CO2uptake once reaching the gate-opening threshold pressure. To better understand this gate-opening mechanism, we investigated its transition process from the perspective of CO2dynamics and its interaction with the framework via variable-temperature13C solid-state nuclear magnetic resonance spectroscopy.Our findings revealed that during the gate-opening process, CO2is initially strongly adsorbed at one site when the gate only slightly opens,while two distinct types of CO2molecules exist when the gate fully opens.11B,13C, and19F magic-angle spinning NMR, in conjunction with in-situ XANES experiments, were also conducted to probethe location of adsorption sites.

Improved solid-state 13C and 15N NMR reveals fundamental compositional divide between refractory dissolved organic carbon and nitrogen in the sea

Marine dissolved organic matter (DOM) is one of the largest reservoirs of organic carbon and nitrogen in the world. Yet, despite its global importance, most DOM remains molecularly uncharacterized. Solid-state nuclear magnetic resonance (NMR) spectroscopy of isolated DOM fractions represents one of the most powerful techniques to understand overall structural composition. However, it is well known that standard cross polarization magic angle spinning (CP/MAS) NMR, the technique used for almost all past solid-state NMR studies of DOM, is at best “semi-quantitative,” and underestimates fully substituted NMR active nuclei. Additionally, almost all past solid-state NMR work analyzed high molecular weight (HMW) material isolated by ultrafiltration, which is now understood to represent mostly 14C-young, “semi-labile” compounds. In contrast, there is far less information regarding the composition of older, low molecular weight (LMW) DOM, which represents the vast majority of the ocean’s accumulated refractory DOM pool.Here, we applied 13C and 15N solid-state multiCP/MAS NMR, improved NMR methods optimized to more quantitatively resolve fully substituted NMR nuclei, to both HMW and LMW DOM isolated from the surface and deep North Pacific Subtropical Gyre. These methods confirm past work indicating most nitrogen containing HMW DOM as amide compounds, but also demonstrate a modest heterocyclic N component not previously identified. In contrast, we found that LMW DON is almost entirely aromatic heterocyclic N, consistent with the hypothesis that heterocyclic N structures may be largely responsible for the accumulation of the ocean’s refractory DON pool. Surprisingly, however, we find DOC aromatic functionalities still represent only a very minor portion of either the HMW or the LMW refractory carbon pools, in marked contrast to refractory DON composition. Together, these more quantitative solid-state NMR techniques likely represent the most accurate picture of DON and DOC functional and compound-class makeup to date, and so have broad implications for our understanding of marine DOM structure and cycling. Specifically, our new data suggests that while chemical composition likely acts as a key control on DOM lability, the most refractory components of DOC and DON have very different compositions, sources, and cycling, supporting the idea that DOC and DON cycling in the ocean may be largely decoupled.

Integrative determination of the atomic structure of mutant huntingtin exon 1 fibrils implicated in Huntington's disease.

Neurodegeneration in Huntington's disease (HD) is accompanied by the aggregation of fragments of the mutant huntingtin protein, a biomarker of disease progression. A particular pathogenic role has been attributed to the aggregation-prone huntingtin exon 1 (HTTex1), generated by aberrant splicing or proteolysis, and containing the expanded polyglutamine (polyQ) segment. Unlike amyloid fibrils from Parkinson's and Alzheimer's diseases, the atomic-level structure of HTTex1 fibrils has remained unknown, limiting diagnostic and treatment efforts. We present and analyze the structure of fibrils formed by polyQ peptides and polyQ-expanded HTTex1 in vitro. Atomic-resolution perspectives are enabled by an integrative analysis and unrestrained all-atom molecular dynamics (MD) simulations incorporating experimental data from electron microscopy (EM), solid-state NMR, and other techniques. Alongside the use of prior data, we report new magic angle spinning NMR studies of glutamine residues of the polyQ fibril core and surface, distinguished via hydrogen-deuterium exchange (HDX). Our study provides a new understanding of the structure of the core as well as surface of aggregated HTTex1, including the fuzzy coat and polyQ-water interface. The obtained data are discussed in context of their implications for understanding the detection of such aggregates (diagnostics) as well as known biological properties of the fibrils.

Elucidating microRNA-34a organisation within human Argonaute-2 by dynamic nuclear polarisation-enhanced magic angle spinning NMR.

Understanding mRNA regulation by microRNA (miR) relies on the structural understanding of the RNA-induced silencing complex (RISC). Here, we elucidate the structural organisation of miR-34a, which is de-regulated in various cancers, in human Argonaute-2 (hAgo2), the effector protein in RISC. This analysis employs guanosine-specific isotopic labelling and dynamic nuclear polarisation (DNP)-enhanced Magic Angle Spinning (MAS) NMR. Homonuclear correlation experiments revealed that the non-A-form helical conformation of miR-34a increases when incorporated into hAgo2 and subsequently bound to SIRT1 mRNA compared to the free miR-34a or the free mRNA:miR duplex. The C8-C1' correlation provided a nucleotide-specific distribution of C2'- and C3'-endo sugar puckering, revealing the capture of diverse dynamic conformations upon freezing. Predominantly C3'-endo puckering was observed for the seed region, while C2'-endo conformation was found in the central region, with a mixture of both conformations elsewhere. These observations provide insights into the molecular dynamics underlying miR-mediated mRNA regulation and demonstrate that experiments conducted under cryogenic conditions, such as at 90K, can capture and reveal frozen dynamic states, using methods like DNP-enhanced MAS NMR or Cryo-ElectronMicroscopy.