NMR Relaxation Studies Research Articles

Structure-activity relationships of ZrO2 crystalline phases in the catalytic transfer hydrogenation of methyl levulinate with ethanol

The depletion of fossil resources is driving the research towards renewable alternatives, like lignocellulosic biomass. Therefore, the development of efficient continuous-flow processes, allowing to achieve better productivity compared to batch processes, will play a crucial role in promoting a sustainable transition. In this context, we report on the continuous-flow, gas-phase, catalytic transfer hydrogenation (CTH) of methyl levulinate and ethanol over zirconia catalysts, in particular focusing on the effect of two different crystalline phases (i.e. monoclinic, m-ZrO2, and tetragonal, t-ZrO2) on catalyst performance. An in-depth catalyst characterisation was coupled with both computational and 1H NMR relaxation studies to assess the structure-activity relationship, providing fundamental insights into the catalytic process and future catalyst optimization. The results, indicate that the higher Lewis acidity and basicity along with the lower affinity with ethanol of m-ZrO2 with respect to t-ZrO2 are responsible for the promotion of undesired oligomerisation reactions of angelica lactones responsible for catalyst deactivation.

Limits of alkanes confined in mesoporous silica as a probe for geometrical tortuosity. An NMR relaxation study

Diffusion Nuclear Magnetic Resonance experiments offer a means to determine the geometrical tortuosity of a porous matrix, assuming that the interaction between the confined fluid and the confining matrix is negligible. In this, alkanes play a crucial role as molecular probes for polar mesoporous systems, with silica being particularly relevant due to its wide range of technological applications. Despite their inherent hydrophobicity, NMR measurements reveal that alkane/silica interactions become significant when the pore diameter approaches the size of the molecules. This fact is clearly demonstrated through the examination of the longitudinal (T1) and transverse (T2) relaxation times of linear and cyclic alkanes confined within silica mesopores of mean pore sizes: 3, 6, and 15 nm. Noticeably, the interactions are practically negligible in 6 and 15 nm pores, but they become relevant in the smallest ones. T1-T2 correlation maps evidence that relaxation times are influenced by molecular length, shape, and pore diameter. These empirical results not only corroborate previous theoretical findings but also highlight the ratio T1/T2 as a valuable, rapid, and cost-effective experimental tool for assessing the feasibility of a given solvent as a molecular probe for determining geometrical tortuosity.

Low-field 2D NMR relaxation and DRIFTS studies of glucose isomerization in zeolite Y: New insights into adsorption effects on catalytic performance

Sn and Ga doped zeolite Y catalysts were tested for the isomerization of glucose to fructose carried out in different solvents (water, methanol and ethanol). Therein, ethanol favoured a Lewis acid site catalyzed pathway that promotes glucose isomerization to fructose, whereas methanol resulted in an equal distribution of products (mannose, fructose and alkyl fructoside). In contrast, the catalysts were totally inactive in water solvent. NMR relaxation measurements, including solvent displacement experiments, suggested that the lack of catalytic activity in water is due to the strong adsorption of this solvent within the zeolite pores blocking reactants from the Lewis acid sites active for the sugar isomerization. In comparison, ethanol adsorbs relatively more strongly than methanol, hence is retained in the pores where solvated fructose is preferentially prevented from the further reaction on Brønsted acid sites situated outside of the pore space. NMR relaxation measurements using pyridine and tetrahydrofuran (THF) and pyridine-DRIFTS measurements suggest metal doping had little effect on the overall relative acid strength of the zeolites but resulted in zeolites with increased Lewis acid strength relative to the non-doped zeolites. The results reported provide direct experimental evidence on the importance of adsorption properties of solvents within zeolites used for glucose to fructose isomerization and may serve as a starting point for a new approach towards designing and optimizing such catalytic systems.

Fast Motions Dominate Dynamics of Intrinsically Disordered Tau Protein at High Temperatures.

Reorientational dynamics of intrinsically disordered proteins (IDPs) contain multiple motions often clustered around three motional modes: ultrafast librational motions of amide groups, fast local backbone conformational fluctuations and slow chain segmental motions. This dynamic picture is mainly based on 15 N NMR relaxation studies of IDPs at relatively low temperatures where the amide-water proton exchange rates are sufficiently small. Less is known, however, about the dynamics of IDPs at more physiological temperatures. Here, we investigate protein dynamics in a 441-residue long IDP, tau protein, in the temperature range from 0-25 °C, using 15 N NMR relaxation rates and spectral density analysis. While at these temperatures relaxation rates are still better described in terms of amide group librational motions, local backbone dynamics and chain segmental motions, the temperature-dependent trend of spectral densities suggests that the timescales of fast backbone conformational fluctuations and slower chain segmental motions might become inseparable at higher temperatures. Our data demonstrate the remarkable dynamic plasticity of this prototypical IDP and highlight the need for dynamic studies of IDPs at multiple temperatures.

Influence of stabilisers on the catalytic activity of supported Au colloidal nanoparticles for the liquid phase oxidation of glucose to glucaric acid: understanding the catalyst performance from NMR relaxation and computational studies

Impact of stabilisers on the catalytic performance of supported Au colloidal nanoparticles for the liquid phase oxidation of glucose to glucaric acid.

The extracellular chaperone clusterin prevents primary and secondary nucleation of an amyloidogenic variant of β2-microglobulin

Amyloid fibril formation by the extracellular protein β2-microglobulin (β2m) and its subsequent accumulation in periarticular tissues have been linked to dialysis-related amyloidosis. A natural variant of human β2m responsible for aggressive systemic amyloidosis contains an aspartate to asparagine mutation at residue 76 (i.e. D76N β2m), which readily forms amyloid fibrils in vitro under physiological conditions. In this study, we examined the role of the extracellular molecular chaperone clusterin in modulating D76N β2m fibril formation in vitro under physiological conditions. The presence of extrinsic charged amino acids modulated D76N β2m fibril formation, implying that electrostatic interactions are involved in the protein’s aggregation. Thioflavin T (ThT) and 1-anilinonaphthalene-8-sulfonate fluorescence assays indicated that clusterin interacts via hydrophobic and electrostatic forces with the monomeric, prefibrillar and fibrillar species of D76N β2m. As a result, clusterin was incorporated into D76N β2m aggregates during the latter’s fibril formation, as indicated by SDS-PAGE of depolymerised fibrils. SYPRO Orange and ThT fluorescence assays suggested that, compared to pure D76N β2m fibrils, those formed in the presence of clusterin are chemically more stable with a reduced ability to act as nucleation seeds. Detailed 15N NMR relaxation studies of mixtures of 15N-labelled β2m with clusterin confirmed that the chaperone interacts transiently and non-specifically with monomeric β2m. Clusterin inhibits both primary and secondary nucleation of D76N β2m fibril formation. In doing so, clusterin binds to D76N β2m fibrils and stabilises them to prevent possible fragmentation. In vivo, the multifaceted chaperone action of clusterin may delay, if not prevent, β2m amyloid proliferation and deposition in tissues.

Applying Crystallography and 19F NMR to investigate dynamics and partner protein interactions in a long chain Flavodoxin

Flavodoxin (Fld) is a small FMN containing protein that is involved in single electron transfer. The long‐chain flavodoxin in Rhodopseudomonas palustris bacteria replaces ferredoxin as a low‐potential electron carrier when iron is scarce. Thus it is proposed to interact with the bifurcating electron transfer flavoprotein (ETF) that yields low‐potential electrons. A surface loop on Fld interacts with another of Fld's partner proteins, so we hypothesize that it also mediates Fld's interaction with ETF. To monitor interactions with ETF directly and investigate dynamics in this loop, we are using 19F NMR in solution. 19F is hyperresponsive to changes in its chemical environment with a chemical shift range of >300 ppm. To provide a static reference point and assess structural heterogeneity, we are also exploiting X‐ray crystallography.In this study we selectively fluorinated the five tyrosine residues in Fld. We obtained resonance assignments from 19F spectra of Fld variants in which individual tyrosine residues have been replaced. We obtain well resolved signals for each residue, but the resonances' linewidths indicate dynamics that affects some resonances more than others. Y90 residue displays two resonances demonstrating two different conformations that interconvert slowly on an NMR time scale. Meanwhile the crystal structure solved at 2.1Å resolution reveals two molecules per asymmetric unit providing two perspectives on the details of the structure. The crystal symmetry is monoclinic in contrast to most of the other Flds, which are orthorhombic. Interestingly, the long loop bearing Y121 and Y123 is not well resolved in chain B of the crystal structure, and the NMR line of Y123 is exceptionally broad, both indicating that the loop is dynamic and capable of altering its conformation to accomodate binding to a partner protein.Future directions include monitoring the changes in the 19F NMR of the Fld when titrated with the partner protein, temperature dependence of the NMR spectrum and relaxation studies to evaluate time scales of motions.

Understanding the Nucleation and Growth of Iron Oxide Nanoparticle Formation by a “Heating-Up” Process: An NMR Relaxation Study

Understanding the Nucleation and Growth of Iron Oxide Nanoparticle Formation by a “Heating-Up” Process: An NMR Relaxation Study

Anhydrous fat crystallization of ultrasonic treated goat milk: DSC and NMR relaxation studies

Methods of NMR relaxation and differential scanning calorimetry (DSC) were used to study the crystallization of anhydrous milk fat (AMF) obtained from milk and subjected to ultrasonic (US) processing. Amongst the changes in the crystallization nature under the influence of ultrasound are the decrease in the crystallization temperature and the increase in the melting enthalpy of the anhydrous milk fat samples. The increase is ∼30% at 20 min of isothermal crystallization and is presumably explained by the additional formation of β'-form crystals from the melt. The parameters of the Avrami equation applied to the description of experimental data show an increase in the crystallization rate in samples with ultrasonic treatment and a change in the dimension of crystallization with a change in melting temperature.

Low-field and variable-field NMR relaxation studies of H2O and D2O molecular dynamics in articular cartilage.

Osteoarthritis (OA) as the main degenerative disease of articular cartilage in joints is accompanied by structural and compositional changes in the tissue. Degeneration is a consequence of a reduction of the amount of macromolecules, the so-called proteoglycans, and of a corresponding increase in water content, both leading to structural weakening of cartilage. NMR investigations of cartilage generally address only the relaxation properties of water. In this study, two-dimensional (T1-T2) measurements of bovine articular cartilage samples were carried out for different stages of hydration, complemented by molecular exchange with D2O and treatment by trypsin which simulates degeneration by OA. Two signal components were identified in all measurements, characterized by very different T2 which suggests liquid-like and solid-like dynamics. These measurements allow the quantification of separate hydrogen components and their assignment to defined physical pools which had been discussed repeatedly in the literature, i.e. bulk-like water and a combination of protein hydrogens and strongly bound water. The first determination of 2H relaxation dispersion in comparison to 1H dispersion suggests intramolecular interactions as the dominating source for the pronounced magnetic field dependence of the longitudinal relaxation time T1.

Dynamics in natural and designed elastins and their relation to elastic fiber structure and recoil

Elastin fibers assemble in the extracellular matrix from the precursor protein tropoelastin and provide the flexibility and spontaneous recoil required for arterial function. Unlike many proteins, a structure-function mechanism for elastin has been elusive. We have performed detailed NMR relaxation studies of the dynamics of the minielastins 24x′ and 20x′ using solution NMR, and of purified bovine elastin fibers in the presence and absence of mechanical stress using solid state NMR. The low sequence complexity of the minielastins enables us to determine average dynamical timescales and degrees of local ordering in the cross-link and hydrophobic modules separately using NMR relaxation by taking advantage of their residue-specific resolution. We find an extremely high degree of disorder, with order parameters for the entirety of the hydrophobic domains near zero, resembling that of simple chemical polymers and less than the order parameters that have been observed in other intrinsically disordered proteins. We find that average backbone order parameters in natural, purified elastin fibers are comparable to those found in 24x′ and 20x′ in solution. The difference in dynamics, compared with the minielastins, is that backbone correlation times are significantly slowed in purified elastin. Moreover, when elastin is mechanically stretched, the high chain disorder in purified elastin is retained, showing that any change in local ordering is below that detectable in our experiment. Combined with our previous finding of a 10-fold increase in the ordering of water when fully hydrated elastin fibers are stretched by 50%, these results support the hypothesis that stretch induced solvent ordering, i.e., the hydrophobic effect, is a key player in the elastic recoil of elastin as opposed to configurational entropy loss.

EPR and NMR study of molecular components mobility and organization in goat milk under ultrasound treatment

The effect of ultrasound treatment on molecular mobility and organization of the main components in raw goat milk was studied by EPR and NMR spectroscopies. NMR relaxation studies showed an increase in the spin–lattice T1 and spin–spin T2 relaxation times in goat milk products (cream, anhydrous fat) and change in the diffusion of proton-containing molecules during ultrasound treatment. The diffusion became more uniform and could be rather accurately approximated by one effective diffusion coefficient Deff, which indicates homogenization of goat milk components, dispersion of globular and supermicellar formations under sonication. EPR studies have shown that molecular mobility and organization of hydrophobic regions in goat milk are similar to those observed in micellar formations of surfactants with a hydrocarbon chain length C12–C16. Ultrasound treatment did not affect submicellar and protein globule organization. Free radicals arising under ultrasound impact of milk reacted quickly with components of goat milk (triglycerides, proteins, fatty acids) and were not observed by spin trapping method.

EPR and NMR Study of Molecular Components Mobility and Organization in Goat Milk Under Ultrasound Treatment

The effect of ultrasound treatment on molecular mobility and organization of the main components in raw goat milk was studied by EPR and NMR spectroscopies. NMR relaxation studies have shown an increase in the spin-lattice T 1 and spin-spin T 2 relaxation times in goat milk products (cream, anhydrous fat) and a change in the nature of diffusion of proton-containing molecules during ultrasound treatment. The diffusion became more uniform and could be rather accurately approximated by one effective diffusion coefficient D eff , which indicates homogenization of goat milk components, dispersion of globular and supermicellar formations under sonication. EPR studies have shown that molecular mobility and organization of hydrophobic regions in goat milk are similar to those observed in micellar formations of surfactants with a hydrocarbon chain length С12-С16. Ultrasound treatment did not affect submicellar and protein globule organization. Free radicals arising under ultrasound impact of milk reacted quickly with components of goat milk (triglycerides, proteins, fatty acids) and were not observed by spin trapping method.

Long-Chain Fatty Acids in Bones and Their Link to Submicroscopic Vascularization Network: NMR Assignment and Relaxation Studies under Magic Angle Spinning Conditions in Intramuscular Bones of Atlantic Herring Fish.

The long-lasting proton signals in bones are identified as long-chain fatty acids, including saturated, mono-, and di-unsaturated fatty acids, with direct nuclear magnetic resonance evidence. We used intramuscular bones from Atlantic Herring fish to avoid interference from lipid-rich marrows. The key is to recognize that these signals are from mobile phase materials and study them with J-coupled correlation spectroscopies under magic angle spinning conditions. We kept extensive 1H-spin-echo records that allowed us to examine the effect of magic angle spinning on the transverse relaxation time of water and lipids over time. While it is impossible to distinguish based on chemical shifts, the relaxation data suggest that the signals are more consistent with the interpretation of phospholipid membranes than triglycerides in lipid droplets. In particular, the simultaneous T2 changes in water and lipids suggest that the centrifugal impact of magic angle spinning alters the lipid's structure in very tight spaces. Additional evidence of phospholipid membranes came from the choline-γ resonance at 3.2 ppm in fresh samples, which disappears with magic angle spinning. Thus, the fatty acid signals are at least partially from membrane bilayer structures, and we propose that they are linked to the submicroscopic vascularization channels similar to the dense canaliculi network in mammalian bones. Our detection of phospholipids from bones depended critically on two factors: (1) the elimination of the overwhelming triglyceride signals from marrows and (2) the preservation of water that biomembranes require. The relaxation data reveal aspects of lipid fluidity that have not been elucidated by previous order parameter studies on model membranes. Relaxation times have long been considered difficult to interpret. A robust and renewed understanding may be beneficial.

Probing Conformational Dynamics by Protein Contact Networks: Comparison with NMR Relaxation Studies and Molecular Dynamics Simulations

Protein contact networks (PCNs) have been used for the study of protein structure and function for the past decade. In PCNs, each amino acid is considered as a node while the contacts among amino acids are the links/edges. We examined the possible correlation between the closeness centrality measure of amino acids within PCNs and their mobility as known from NMR spin relaxation experiments and molecular dynamic (MD) simulations. The pivotal observation was that plasticity within a protein stretch correlated inversely to closeness centrality. Effects on protein conformational plasticity caused by the formation of disulfide bonds or protein–protein interactions were also identified by the PCN analysis measure closeness centrality and the hereby introduced percentage of closeness centrality perturbation (% CCP). All the comparisons between PCN measures, NMR data, and MDs were performed in a set of proteins of different biological functions and structures: the core protease domain of anthrax lethal factor, the N-terminal RING domain of E3 Ub ligase Arkadia, the reduced and oxidized forms of human thioredoxin 1, and the ubiquitin molecules (Ub) of the catalytic Ub–RING–E3–E2–Ub complex of E3 ligase Ark2.The graph theory analysis of PCNs could thus provide a general method for assessing the conformational dynamics of free proteins and putative plasticity changes between different protein forms (apo/complexed or reduced/oxidized).

Structural Studies of the Interaction Between the Paddle Motif from NaV1.5Div and the Sea Anemone Toxin Anthopleurin-A

Voltage gated sodium channels (VGSC) are membrane proteins that are important in the central and peripheral nervous systems and cardiac muscles among others. The main function of VGSC is in the propagation of electrical signals by depolarizing excitable cells. Numerous diseases have been linked to defects in VGSC including epilepsy, mental retardation, and several ailments that could lead to sudden cardiac death. Furthermore, these channels are one of the primary targets of toxins from venomous animals. These toxins have been used as an excellent probe to study the function of VGSC due to their specific and potent effect on VGSC. The sea anemone toxin anthopleurin-A (ApA) inhibits the fast inactivation of the cardiac channel NaV1.5 by binding to the S3b-S4a motif (paddle motif) on the voltage sensing domain (VSD) of DIV. Here we used NMR spectroscopy to determine the solution structures of NaV1.5 DIV paddle motif and of ApA toxin, both in DPC micelles. The structure of the paddle motif takes a helix-turn-helix motif that aligns well with the cryo-EM structure of the full channel. Through paramagnetic relaxation enhancement, we determined that the paddle motif is mainly interacting with the interface region of the micelle. Based on 15N NMR relaxation studies, the paddle motif is mostly rigid with some residues at and around the loop in addition to the C-terminus undergoing ps-ns motion. NMR titration experiments between a 15N labeled paddle motif with unlabeled ApA toxin uncovered specific residues on the paddle that likely interact with the toxin. Further electrophysiology studies are needed to confirm the importance of these residues in toxin binding. We are in the process of determining the three-dimensional structure of the toxin-paddle complex in micelle.

Membrane Deformation Modulated by Hydration and Cholesterol Unveiled by Solid-State 2H NMR Spectroscopy

Phospholipid membranes are biological liquid crystals in which intermolecular interactions at mesoscopic length scales play key roles in the emergence of membrane physical properties like structure and rigidity. Such interactions modulated by membrane hydration [1] and cholesterol [2] have been investigated using solid-state 2H NMR spectroscopy. The average structure is manifested by the lineshapes through principal values of static or residual coupling tensors due to quadrupolar interactions. The residual quadrupolar couplings (RQCs) yield segmental order parameters for the individual acyl segments (i), providing average membrane properties such as bilayer thickness, area/lipid, and spontaneous curvature [3]. We show that membrane dehydration and increased cholesterol content result in greater RQCs, indicative of membrane thickening. Elastic membrane deformations are exhibited as collective order-director fluctuations (ODF) contributing to the nuclear magnetic relaxation. At mesoscopic length scales the thermal fluctuations connect to membrane mechanical properties via the fluctuation-dissipation theorem. Model-free interpretation of the functional dependence of spin-lattice relaxation rates, R1Z, on segmental order parameters (square-law) reveals emergent material properties in the liquid-crystalline state. The R1Z relaxation dispersion and temperature dependence of square-law plots indicate a nematic-like membrane hydrocarbon core. Solid-state 2H NMR relaxation studies demonstrate that both dehydration and increased cholesterol content in the membrane lead to decreased slopes of square-law plots, indicating an increase in membrane bending rigidity, consistent with neutron spin-echo (NSE) measurements. Analogous 2H NMR studies with various membrane lipid compositions and peptide-reconstituted membranes have addressed lipid-mediated cell signaling and lipid-protein interactions. Molecular dynamics simulations provide further insights on the correspondence of the solid-state NMR and NSE results. [1] K.J. Mallikarjunaiah et al. (2019) Phys. Chem. Chem. Phys. 21, 18422. [2] S. Chakraborty et al. (2020) Proc. Natl. Acad. Sci. 117, 21896. [3] Mallikarjunaiah et al. (2011) Biophys. J. 100, 98.

The influence of extruded flour on water content and retrogradation process in muffins during storage: NMR relaxation study.

A comparative study of the moisture content, mobility of water protons and retrogradation process in muffin samples with the addition of extruded (ME) flour (10%) during storage (14days) was carried out and compared to control samples without the addition (MO). A significant increase in water content during swelling of the extruded flour was found. The proton magnetic relaxation signal for both ME and MO showed a two-component character for the fast-decay region (free induction decay, FID experiment) and a three-component character for the slow-decay (CPMG experiment) one. The relaxation times T2 characterizing the protons of both water and non-aqueous fraction decreased during the experiment except for the protons of the fat fraction. The values of relaxation times T2 for ME exceeded the values for MO, which reflects the increased water content in ME. The amplitude of the signal of water protons (two components of CPMG) decreased for ME more slowly (by about 3-5days), which can serve as a characterization the shelf life of the muffins. Estimation of the retrogradation rate according to the Avrami model showed a lower value of the rate for ME compared to MO, which may reflect the increased moisture content in ME.

Assessing the Direct Binding of Ark-Like E3 RING Ligases to Ubiquitin and Its Implication on Their Protein Interaction Network.

The ubiquitin pathway required for most proteins’ targeted degradation involves three classes of enzymes: E1-activating enzyme, E2-conjugating enzyme, and E3-ligases. The human Ark2C is the single known E3 ligase that adopts an alternative, Ub-dependent mechanism for the activation of Ub transfer in the pathway. Its RING domain binds both E2-Ub and free Ub with high affinity, resulting in a catalytic active UbR-RING-E2-UbD complex formation. We examined potential changes in the conformational plasticity of the Ark2C RING domain and its ligands in their complexed form within the ubiquitin pathway through molecular dynamics (MD). Three molecular mechanics force fields compared to previous NMR relaxation studies of RING domain of Arkadia were used for effective and accurate assessment of MDs. Our results suggest the Ark2C Ub-RING docking site has a substantial impact on maintaining the conformational rigidity of E2-E3 assembly, necessary for the E3’s catalytic activity. In the UbR-RING-E2-UbD catalytic complex, the UbR molecule was found to have greater mobility than the other Ub, bound to E2. Furthermore, network-based bioinformatics helped us identify E3 RING ligase candidates which potentially exhibit similar structural modules as Ark2C, along with predicted substrates targeted by the Ub-binding RING Ark2C. Our findings could trigger a further exploration of related unrevealed functions of various other E3 RING ligases.

Mimicking the graphene oxide structure in solutions by interaction of Fe(III) and Gd(III) with model small-size ligands. The NMR relaxation study

Despite long history and recent progress, the chemical behavior of graphene oxide (GO) is not fully understood. One of the remaining open questions is an extremely high cation exchange capacity, which cannot be explained in the frames of the commonly accepted Lerf-Klinowski structural model. The interaction of GO with metals, termed as sorption, is usually considered as physisorption, or as non-specific electrostatic attraction. Recently, we demonstrated that in fact this interaction has the coordinate-covalent bonding nature. In this work, by the means of the NMR relaxation, we investigated the interaction of Fe(III) and Gd(III) ions with several small molecule size chelators and with linear polymers in order to possibly mimic the GO structure. The experiments were conducted in the broad pH range, and at the different metal/chelator ratios. From the two tested metal ions, Fe(III) has the stronger affinity toward GO due to its higher charge density. GO binds Fe(III) with formation of strong high-relaxivity complexes, which do not undergo hydrolysis even in basic conditions. Numerous model small-size molecules, tested to mimic the GO structure, did not demonstrate the same effect on the relaxation of the Fe(III) and Gd(III) solutions, as does interaction with GO. From the tested linear polymers, only polyacrylic acid (PAA) can bind Fe(III), however, even PAA, consisting from carboxyl groups, cannot prevent Fe(III) from hydrolysis already in neutral solutions. Subsequently, carboxyl groups were excluded from the species in GO structure, responsible for the strong binding of meatal ions. We conclude that there are no preexisting sites or functional group fragments in GO structure, capable to strongly bind transition metal cations. The binding sites are formed from the existing structure upon the reaction with metal cations, to afford formation of the metal/GO coordination compounds. The most probable change in the GO structure is the formation of enolates from tertiary alcohols with rupture of the CC bonds. The newly reported data provide additional experimental evidence for the Dynamic Structural Model of GO and broadens the areas of its applicability.