When Li-saturated montmorillonite is heated to 200–300 °C, the Li ions migrate from interlayer positions to sites in the layer structure. However, the identity of these sites has not been clearly established. Here we have investigated the migration of Li ions in montmorillonite, after heat treatment at 250 °C, using chemical and instrumental analyses. The latter include X-ray diffractometry (XRD), 7Linuclear magnetic resonance (NMR) spectroscopy and X-ray photoelectron spectroscopy (XPS). Heating causes a large reduction in cation exchange capacity (CEC) and an almost complete loss of interlayer expansion with glycerol as shown by XRD. Static and magic angle spinning (MAS) 7Li-NMR spectroscopy shows that the quadrupole coupling constant of Li increases markedly over the corresponding value for unheated Li-montmorillonite (where Li occupies exchange sites in the interlayer space) and for hectorite (where Li is located in the octahedral sheet). This would indicate that, in heated montmorillonite, Li occupies structural sites of low symmetry which, however, cannot be identified with octahedral vacancies in the layer structure as is commonly assumed. XPS shows that the binding energy (BE) for Li in unheated montmorillonite is comparable to that for other exchangeable cations. Heating broadens the Li 1s band and decreases the BE. The BE for Li in heated montmorillonite is significantly higher than that in either spodumene or lepidolite, where Li is known to occupy octahedral sites. The combined data suggest that heating induces Li to migrate from interlayer sites to ditrigonal cavities in the tetrahedral sheet, rather than into vacancies in the octahedral sheet, of montmorillonite.

Samples of Silica Springs allophane from Tongariro National Park, New Zealand, having Al/Si atomic ratios in the range 1.1-1.9, were studied by 27Al nuclear magnetic resonance (NMR) spectroscopy with high field strength (9.4 and 11.7 T) and fast magic-angle spinning (MAS) (9-13 kHz). Spectra for all samples show peaks for 6- and 4-coordinate Al and also for 5-coordinate Al. For 1 sample, the peak for 5-coordinate Al is dominant. Use of 2 instruments and 2 field strengths allowed the integrity of the spectra and the assignment of 5-coordinate Al to be verified. The “true” chemical shift (after a small correction for quadrupolar shift) observed for 5-coordinate Al in Silica Springs allophane is 36 ± 1 ppm, which is consistent with shifts reported for 5-coordination in well-characterized crystalline structures. We suggest that 5-coordination in Silica Springs allophane is associated with the edges of fragments of incomplete octahedral sheets that are bonded to disordered, though more complete, curved tetrahedral sheets in the primary particles of this allophane. Other allophanes with Al/Si < 2, and which are poor in octahedra relative to tetrahedra, may also have significant Al in 5-coordinate sites.

Cardiac hypertrophy (CH) is an augmentation of either the right ventricular or the left ventricular mass in order to compensate for the increase of work load on the heart. Metabolic abnormalities lead to histological changes of cardiac myocytes and turn into CH. The molecular mechanisms that lead to initiate CH have been of widespread concern, hence the development of the new field of research, metabolomics: one ‘omics’ approach that can reveal comprehensive information of the paradigm shift of metabolic pathways network in contrast to individual enzymatic reaction-based metabolites, have attempted and until now only 19 studies have been conducted using experimental animal and human specimens. Nuclear magnetic resonance spectroscopy and mass spectrometry-based metabolomics studies have found that CH is a metabolic disease and is mainly linked to the harmonic imbalance of glycolysis, citric acid cycle, amino acids and lipid metabolism. The current review will summarise the main outcomes of the above mentioned 19 studies that have expanded our understanding of the molecular mechanisms that may lead to CH and eventually to heart failure.

SARS-CoV-2 nucleocapsid (N) protein plays the essential roles in key steps of the viral life cycle, thus representing a top drug target. Functionality of N protein including liquid–liquid phase separation (LLPS) depends on its interaction with nucleic acids. Only the variants with N proteins functional in binding nucleic acids might survive and spread in evolution and indeed, the residues critical for binding nucleic acids are highly conserved. Hydroxychloroquine (HCQ) was shown to prevent the transmission in a large-scale clinical study in Singapore but so far, no specific SARS-CoV-2 protein was experimentally identified to be targeted by HCQ. Here by NMR, we unambiguously decode that HCQ specifically binds NTD and CTD of N protein with Kd of 112.1 and 57.1 μM, respectively to inhibit their interaction with nucleic acid, as well as to disrupt LLPS. Most importantly, HCQ-binding residues are identical in SARS-CoV-2 variants and therefore HCQ is likely effective to different variants. The results not only provide a structural basis for the anti-SARS-CoV-2 activity of HCQ, but also renders HCQ to be the first known drug capable of targeting LLPS. Furthermore, the unique structure of the HCQ-CTD complex suggests a promising strategy for design of better anti-SARS-CoV-2 drugs from HCQ.

In vivo NMR 31p spectroscopy is a non invasive, non ionizing method of exploration of energy and phospholipid metabolism in the brain. This study consisted of comparing 31p spectra in five patients with Senile Dementia of Alzheimer Type (SDAT) with those of four controls of similar ages. Abnormal phosphonionocsters (PME) concentrations, either high or low, were found in the patients, but statistical analysis did not elicit any significant difference relative to controls.

Spontaneous deamidation prompted backbone isomerization of Asn/Asp residues resulting in – most cases – the insertion of an extra methylene group into the backbone poses a threat to the structural integrity of proteins. Here we present a systematical analysis of how temperature, pH, presence of charged residues, but most importantly backbone conformation and dynamics affect isomerization rates as determined by nuclear magnetic resonance in the case of designed peptide-models. We demonstrate that restricted mobility (such as being part of a secondary structural element) may safeguard against isomerization, but this protective factor is most effective in the case of off-pathway folds which can slow the reaction by several magnitudes compared to their on-pathway counterparts. We show that the geometric descriptors of the initial nucleophilic attack of the isomerization can be used to classify local conformation and contribute to the design of stable protein drugs, antibodies or the assessment of the severity of mutations.

The objective of the present explorative study was to determine the absorption dynamics when feeding diets varying in types and levels of dietary fibre in a catheterised animal model. A total of six sows were fed a diet low in fibre (LF), a diet high in soluble fibre and a diet high in insoluble fibre in a repeated 3 × 3 cross-over design. Plasma samples were collected from the mesenteric artery and the portal vein to determine different absorption phases by 1H NMR spectroscopy-based metabonomics. Time profiles were determined for plasma levels of specific metabolites and for the absorption of these metabolites from the small intestine. The LF diet resulted in a higher betaine concentration in the blood than the two high-fibre diets (P = 0·008). This leads to higher plasma concentrations of methionine (P = 0·0028) and creatine (P = 0·020) of endogenous origin. In conclusion, the use of NMR spectroscopy for measuring nutrient uptake in the present study elucidated the relationship between betaine uptake and elevated creatine plasma concentrations.

The polyphagous insect Sitophilus oryzae L. (Coleoptera:Curculionidae) has a tremendous adaptability in feeding behaviour, making it a serious invasive pest of stored cereals. The present study identifies the metabolite composition of Sitophilus oryzae (S. oryzae) using Nuclear Magnetic Resonance (NMR) spectroscopy. Assignment of 1D-proton by NMR, 1H-1H COSY, 2D-TOCSY 1H-1H, had been done. Amongst the various biochemically important metabolites isoleucine, valine, leucine, β-hydroxybutyrate, lysine, glutamate, glutamine, proline, lactate, alanine, di-methylamine, α-glucose, β-glucose, choline, glycerophosphorylcholine and tyrosine are present in S. oryzae. In wheat-fed S. oryzae, the presence of threonine and the absence of lactate is observed. In rice-fed S. oryzae, however, the presence of lactate and the absence of threonine were observed. Barley-fed S. oryzae shows presence of both tyrosine and lactate. It is concluded that the pest S. oryzae has adaptability on different stored cereals and grains, depicting the presence of earlier reported metabolites. The present study aims to identify the key metabolic components and associated enzymes in Sitophilus oryzae fed on different cereals.

Polysaccharides from thalli of Lasallia pustulata were extracted and characterized. The major fractions were characterized as pustulan, a β-glucan-chitin complex and a β-(1→3)-glucan. Other polysaccharides, which are minor components (less than 5%) solubilized with water from the alkali extracts of thalli, were shown to be galactomannans with a backbone of α-(l→6)- mannose partially substituted at positions 2 by galactofuranose and/or mannopyranose side chains of variable length. The relationship between these polysaccharides and those obtained by the same method from free-living fungi is discussed. The proportion or composition of the different polysaccharides studied was not affected by seasonal environmental conditions over a 2-year period.

The three-dimensional structure of the anti-apoptotic protein Bcl-xL complexed to a 25-residue peptide from the death promoting region of Bad was determined using NMR spectroscopy. Although the overall structure is similar to Bcl-xL bound to a 16-residue peptide from the Bak protein (Sattler et al., 1997), the Bad peptide forms additional interactions with Bcl-xL. However, based upon site-directed mutagenesis experiments, these additional contacts do not account for the increased affinity of the Bad 25-mer for Bcl-xL compared to the Bad 16-mer. Rather, the increased helix propensity of the Bad 25-mer is primarily responsible for its greater affinity for Bcl-xL. Based on this observation, a pair of 16-residue peptides were designed and synthesized that were predicted to have a high helix propensity while maintaining the interactions important for complexation with Bcl-xL. Both peptides showed an increase in helix propensity compared to the wild-type and exhibited an enhanced affinity for Bcl-xL.

The ligand-binding domain of the human low-density lipoprotein receptor consists of seven modules, each of 40–45 residues. In the presence of calcium, these modules adopt a common polypeptide fold with three conserved disulfide bonds. A concatemer of the first and second modules (LB1–2) folds efficiently in the presence of calcium ions, forming the same disulfide connectivities as in the isolated modules. The three-dimensional structure of LB1–2 has now been solved using two-dimensional 1H NMR spectroscopy and restrained molecular dynamics calculations. No intermodule nuclear Overhauser effects were observed, indicating the absence of persistent interaction between them. The near random-coil NH and Hα chemical shifts and the low φ and ψ angle order parameters of the four-residue linker suggest that it has considerable flexibility. The family of LB1–2 structures superimposed well over LB1 or LB2, but not over both modules simultaneously. LB1 and LB2 have a similar pattern of calcium ligands, but the orientations of the indole rings of the tryptophan residues W23 and W66 differ, with the latter limiting solvent access to the calcium ion. From these studies, it appears that although most of the modules in the ligand-binding region of the receptor are joined by short segments, these linkers may impart considerable flexibility on this region.

Within the tumor suppressor protein INK4 (inhibitor of cyclin-dependent kinase 4) family, p15INK4B is the smallest and the only one whose structure has not been determined previously, probably due to the protein's conformational flexibility and instability. In this work, multidimensional NMR studies were performed on this protein. The first tertiary structure was built by comparative modeling with p16INK4A as the template, followed by restrained energy minimization with NMR constraints (NOE and H-bonds). For this purpose, the solution structure of p16INK4A, whose quality was also limited by similar problems, was refined with additional NMR experiments conducted on an 800 MHz spectrometer and by structure-based iterative NOE assignments. The nonhelical regions showed major improvement with root-mean-square deviation (RMSD) improved from 1.23 to 0.68 Å for backbone heavy atoms. The completion of p15INK4B coupled with refinement of p16INK4A made it possible to compare the structures of the four INK4 members in depth, and to compare the structures of p16INK4A in the free form and in the p16INK4A-CDK6 complex. This is an important step toward a comprehensive understanding of the precise functional roles of each INK4 member.

Helical coiled-coils and bundles are some of the most common structural motifs found in proteins. Design and synthesis of α-helical motifs may provide interesting scaffolds that can be useful as host structures to display functional sites, thus allowing the engineering of novel functional miniproteins. We have synthesized a 38-amino acid peptide, α2p8, encompassing the α-helical hairpin present in the structure of p8MTCP1, as an α-helical scaffold particularly promising for its stability and permissiveness of sequence mutations. The three-dimensional structure of this peptide has been solved using homonuclear two-dimensional NMR techniques at 600 MHz. After sequence specific assignment, a total of 285 distance and 29 dihedral restraints were collected. The solution structure of α2p8 is presented as a set of 30 DIANA structures, further refined by restrained molecular dynamics, using simulated annealing protocol with the AMBER force field. The RMSD values for the backbone and all heavy atoms are 0.65 ± 0.25 and 1.51 ± 0.21 Å, respectively. Excised from its protein context, the α-hairpin keeps its native structure: an α-helical coiled-coil, similar to that found in superhelical structures, with two helices spanning residues 4–16 and 25–36, and linked by a short loop. This motif is stabilized by two interhelical disulfide bridges and several hydrophobic interactions at the helix interface, leaving most of its solvent-exposed surface available for mutation. This α-helical hairpin, easily amenable to synthetic chemistry and biological expression system, may represent a stable and versatile scaffold to display new functional sites and peptide libraries.

The pressure-induced changes in 15N enriched HPr from Staphylococcus carnosus were investigated by two-dimensional (2D) heteronuclear NMR spectroscopy at pressures ranging from atmospheric pressure up to 200 MPa. The NMR experiments allowed the simultaneous observation of the backbone and side-chain amide protons and nitrogens. Most of the resonances shift downfield with increasing pressure indicating generalized pressure-induced conformational changes. The average pressure-induced shifts for amide protons and nitrogens are 0.285 ppm GPa−1 at 278 K and 2.20 ppm GPa−1, respectively. At 298 K the corresponding values are 0.275 and 2.41 ppm GPa−1. Proton and nitrogen pressure coefficients show a significant but rather small correlation (0.31) if determined for all amide resonances. When restricting the analysis to amide groups in the β-pleated sheet, the correlation between these coefficients is with 0.59 significantly higher. As already described for other proteins, the amide proton pressure coefficients are strongly correlated to the corresponding hydrogen bond distances, and thus are indicators for the pressure-induced changes of the hydrogen bond lengths. The nitrogen shift changes appear to sense other physical phenomena such as changes of the local backbone conformation as well. Interpretation of the pressure-induced shifts in terms of structural changes in the HPr protein suggests the following picture: the four-stranded β-pleated sheet of HPr protein is the least compressible part of the structure showing only small pressure effects. The two long helices a and c show intermediary effects that could be explained by a higher compressibility and a concomitant bending of the helices. The largest pressure coefficients are found in the active center region around His15 and in the regulatory helix b which includes the phosphorylation site Ser46 for the HPr kinase. This suggests that this part of the structure occurs in a number of different structural states whose equilibrium populations are shifted by pressure. In contrast to the surrounding residues of the active center loop that show large pressure effects, Ile14 has a very small proton and nitrogen pressure coefficient. It could represent some kind of anchoring point of the active center loop that holds it in the right place in space, whereas other parts of the loop adapt themselves to changing external conditions.

A 20-residue peptide E5 containing five glutamates, an analog of the fusion peptide of influenza virus hemagglutinin (HA) exhibiting fusion activity at acidic pH lower than 6.0–6.5 was studied by circular dichroism (CD), Fourier transform infrared, and 1H-NMR spectroscopy in water, water/trifluoroethanol (TFE) mixtures, dodecylphosphocholine (DPC) micelles, and phospholipid vesicles. E5 became structurally ordered at pH ≤6 and the helical content in the peptide increased in the row: water < water/TFE < DPC ∼ phospholipid vesicle while the amount of β-structure was approximately reverse. 1H-NMR data and line-broadening effect of 5-, 16-doxylstearates on proton resonances of DPC bound peptide showed E5 forms amphiphilic α-helix in residues 2–18, which is flexible in 11–18 part. The analysis of the proton chemical shifts of DPC bound and CD intensity at 220 nm of phospholipid bound E5 showed that the pH dependence of helical content is characterized by the same pKa ≈5.6. Only Glu11 and Glu15 in DPC bound peptide showed such elevated pKas, presumably due to transient hydrogen bond(s) Glu11 (Glu15) δCOO−(H+) … HN Glu15 that dispose(s) the side chain of Glu11 (Glu15) residue(s) close to the micelle/water interface. These glutamates are present in the HA-fusion peptide and the experimental half-maximal pH of fusion for HA and E5 peptides is ∼5.6. Therefore, a specific anchorage of these peptides onto membrane necessary for fusion is likely driven by the protonation of the carboxylate group of Glu11 (Glu15) residue(s) participating in transient hydrogen bond(s).

Circular dichroism and NMR spectroscopy have been used to determine the structure of the low-density lipoprotein (LDL) receptor-binding peptide, comprising residues 130–152, of the human apolipoprotein E. This peptide has little persistent three-dimensional structure in solution, but when bound to micelles of dodecylphosphocholine (DPC) it adopts a predominantly α-helical structure. The three-dimensional structure of the DPC-bound peptide has been determined by using 1H-NMR spectroscopy: the structure derived from NOE-based distance constraints and restrained molecular dynamics is largely helical. The derived φ and ψ angle order parameters show that the helical structure is well defined but with some flexibility that causes the structures not to be superimposable over the full peptide length. Deuterium exchange experiments suggest that many peptide amide groups are readily accessible to the solvent, but those associated with hydrophobic residues exchange more slowly, and this helix is thus likely to be positioned on the surface of the DPC micelles. In this conformation the peptide has one hydrophobic face and two that are rich in basic amino acid side chains. The solvent-exposed face of the peptide contains residues previously shown to be involved in binding to the LDL receptor.

Three peptides covering the sequence regions corresponding to the first two (CspB-1), the first three (CspB-2), and the last two (CspB-3) β-strands of CspB, the major cold shock protein of Bacillus subtilis, have been synthesized and analyzed for their conformations in solution and for their precipitation behavior. The peptides are nearly insoluble in water, but highly soluble in aqueous solutions containing 50% acetonitrile (pH 4.0). Upon shifts of the solvent condition toward lower or higher acetonitrile concentrations, the peptides all form fibrils resembling those observed in amyloid associated diseases. These fibrils have been identified and characterized by electron microscopy, binding of the dye congo red, and X-ray fiber diffraction. Characterization of the peptides in solution by circular dichroism and NMR spectroscopy shows that the formation of these fibrils does not require specific preformed secondary structure in the solution state species. While the majority of the soluble fraction of each peptide is monomeric and unstructured, different types of structures including α-helical, β-sheet, and random coil conformations are observed under conditions that eventually lead to fibril formation. We conclude that the absence of tertiary contacts under solution conditions where binding interactions between peptide units are still favorable is a crucial requirement for amyloid formation. Thus, fragmentation of a sequence, like partial chemical denaturation or mutation, can enhance the capacity of specific protein sequences to form such fibrils.

This paper reports on an insulin analogue with 12.5-fold receptor affinity, the highest increase observed for a single replacement, and on its solution structure, determined by NMR spectroscopy.

The analogue is [d-AlaB26]des-(B27–B30)-tetrapeptide-insulin-B26-amide. C-terminal truncation of the B-chain by four (or five) residues is known not to affect the functional properties of insulin, provided the new carboxylate charge is neutralized. As opposed to the dramatic increase in receptor affinity caused by the substitution of d-Ala for the wild-type residue TyrB26 in the truncated molecule, this very substitution reduces it to only 18% of that of the wild-type hormone when the B-chain is present in full length.

The insulin molecule in solution is visualized as an ensemble of conformers interrelated by a dynamic equilibrium. The question is whether the “active” conformation of the hormone, sought after in innumerable structure/function studies, is or is not included in the accessible conformational space, so that it could be adopted also in the absence of the receptor. If there were any chance for the active conformation, or at least a predisposed state to be populated to a detectable extent, this chance should be best in the case of a superpotent analogue. This was the motivation for the determination of the three-dimensional structure of [d-AlaB26]des-(B27–B30)-tetrapeptide-insulin-B26-amide. However, neither the NMR data nor CD spectroscopic comparison of a number of related analogues provided a clue concerning structural features predisposing insulin to high receptor affinity. After the present study it seems more likely than before that insulin will adopt its active conformation only when exposed to the force field of the receptor surface.