Milligram-scale assembly and NMR fingerprint of tau fibrils adopting the Alzheimer's disease fold.

In the Alzheimer's disease (AD) brain, the microtubule-associated protein tau aggregates into paired helical filaments in which each protofilament has a C-shaped conformation. In vitro assembly of tau fibrils adopting this fold is highly valuable for both fundamental and applied studies of AD without requiring patient-brain extracted fibrils. To date, reported methods for forming AD-fold tau fibrils have been irreproducible and sensitive to subtle variations in fibrillization conditions. Here, we describe a route to reproducibly assemble tau fibrils adopting the AD fold on the multi-milligram scale. We investigated the fibrillization conditions of two constructs and found that a tau (297-407) construct that contains four AD phospho-mimetic glutamate mutations robustly formed the C-shaped conformation. 2D and 3D correlation solid-state NMR spectra show a single predominant set of chemical shifts, indicating a single molecular conformation. Negative-stain electron microscopy and cryo-EM data confirm that the protofilament formed by 4E-tau (297-407) adopts the C-shaped conformation, which associates into paired, triple, and quadruple helical filaments. In comparison, NMR spectra indicate that a previously reported construct, tau (297-391), forms a mixture of a four-layered dimer structure and the C-shaped structure, whose populations are sensitive to the environmental conditions. The determination of the NMR chemical shifts of the AD-fold tau opens the possibility for future studies of tau fibril conformations and ligand binding by NMR. The quantitative assembly of tau fibrils adopting the AD fold should facilitate the development of diagnostic and therapeutic compounds that target AD tau.

A multi-descriptor analysis of substituent effects on the structure and aromaticity of benzene derivatives: π-Conjugation versus charge effects.

This work provides a detailed multi-component analysis of aromaticity in monosubstituted (X = CH3, C H 2 - , C H 2 + , NH2, NH-, NH+, OH, O-, and O+) and para-homodisubstituted (X = CH3, CH2, NH2, NH, OH, and O) benzene derivatives. We investigate the effects of substituents using single-reference (B3LYP/DFT) and multireference (CASSCF/MRCI) methods, focusing on structural (HOMA), vibrational (AI(vib)), topological (ELFπ), electronic (MCI), magnetic (NICS), and stability (S0-T1 splitting) properties. The findings reveal that appropriate π-electron-donating and π-electron-accepting substituents with suitable size and symmetry can interact with the π-system of the ring, significantly influencing π-electron delocalization. While the charge factor has a minimal impact on π-electron delocalization, the presence of a pz orbital capable of interacting with the π-electron delocalization is the primary factor leading to a deviation from the typical aromaticity characteristics observed in benzene.

Aromaticity in the Electronic Ground and Lowest Triplet States of Molecules with Fused Thiophene Rings.

Analysis of the variations of the off-nucleus isotropic magnetic shielding, σiso(r), around thiophene, thienothiophenes, dithienothiophenes and sulflowers in their electronic ground (S0) and lowest triplet (T1) states reveals that some of the features of aromaticity and bonding in these molecules do not fit in with predictions based on the popular Hückel's and Baird's rules. Despite having 4n π electrons, the S0 states of the sulflowers are shown to be aromatic, due to the local aromaticities of the individual thiophene rings. To reduce its T1 antiaromaticity, the geometry of thiophene changes considerably between S0 and T1: In addition to losing planarity, the carbon-carbon two 'double' and one 'single' bonds in S0 turn into two 'single' and one 'double' bonds in T1. Well-defined Baird-style aromaticity reversals are observed between the S0 and T1 states of only three of the twelve thiophene-based compounds investigated in this work, in contrast, the sulflower with six thiophene rings which is weakly aromatic in S0 becomes more aromatic in T1. The results suggest that the change in aromaticity between the S0 and T1 states in longer chains of fused rings is likely to affect mostly the central ring (or the pair of central rings); rings sufficiently far away from the central ring(s) can retain aromatic character.

Analysis of coordinated NMR chemical shifts to map allosteric regulatory networks in proteins.

The exquisite sensitivity of the NMR chemical shift to local environment makes it an ideal probe to assess atomic level perturbations in proteins of all sizes and structural compositions. Recent advances in solution and solid-state NMR spectroscopy of biomolecules have leveraged the chemical shift to report on short- and long-range couplings between individual amino acids to establish "networks" of residues that form the basis of allosteric pathways that transmit chemical signals through the protein matrix to induce functional responses. The simple premise that thermodynamically and functionally coupled regions of a protein (i.e. active and allosteric sites) should be reciprocally sensitive to structural or dynamic perturbations has enabled NMR spectroscopy, the premier method for molecular resolution of protein structural fluctuations, to occupy a place at the forefront of investigations into protein allostery. Here, we detail several key methods of NMR chemical shift analysis to extract mechanistic information about long-range chemical signaling in a protein, focusing on practical methodological aspects and the circumstances under which a given approach would be relevant. We also detail some of the experimental considerations that should be made when applying these methods to specific protein systems.

On the geometry dependence of the nuclear magnetic resonance chemical shift of mercury in thiolate complexes: A relativistic density functional theory study.

Thiolate containing mercury(II) complexes of the general formula [Hg(SR) n ] 2 - n have been of great interest since the toxicity of mercury was recognized. 199Hg nuclear magnetic resonance spectroscopy (NMR) is a powerful tool for characterization of mercury complexes. In this work, the Hg shielding constants in a series of [Hg(SR) n ] 2 - n complexes are therefore investigated computationally with particular emphasis on their geometry dependence. Geometry optimizations and NMR chemical shift calculations are performed at the density functional theory (DFT) level with both the zeroth-order regular approximation (ZORA) and four-component relativistic methods. The four exchange-correlation (XC) functionals PBE0, PBE, B3LYP, and BLYP are used in combination with either Dyall's Gaussian-type (GTO) or Slater-type orbitals (STOs) basis sets. Comparing ZORA and four-component calculations, one observes that the calculated shielding constants for a given molecular geometry have a constant difference of ∼ 1070 ppm. This confirms that ZORA is an acceptable relativistic method to compute NMR chemical shifts. The combinations of four-component/PBE0/v3z and ZORA/PBE0/QZ4P are applied to explore the geometry dependence of the isotropic shielding. For a given coordination number, the distance between mercury and sulfur is the key factor affecting the shielding constant, while changes in bond and dihedral angles and even different side groups have relatively little impact.

Acetonyl C

This paper presents the first example of the formation of acetonyl tridentate CˆNˆN complexes of arylbipyridines in the reaction of chloroplatinum complexes with acetone in the presence of alkali. The chemical structure of obtained substances was established by means of 1H,13C NMR, COSY, HSQC, and HMBC techniques. The attribution of all proton and carbon signals in NMR spectra was performed using 1D and 2D NMR experiments for the synthesized acetonyl cycloplatinated complexes. A comparative analysis of the values of the C-Pt spin-spin coupling constants of the same order was carried out, which showed a significant difference in bond lengths and valence angles inthe cyclic fragments of the arylbipyridine ligand.

1H and 13C NMR spectra of infinitene and the ring current effect of the aromatic molecule.

The spatial magnetic properties (through-space NMR shieldings-TSNMRSs-actually the ring current effect in 1H NMR spectroscopy) of the recently synthesized infinitene (the helically twisted [12]circulene) have been calculated using the GIAO perturbation method employing the nucleus-independent chemical shift (NICS) concept and visualized as iso-chemical-shielding surfaces (ICSS) of various size and direction. Both 1H and 13C chemical shifts of infinitene and the aromaticity of this esthetically very appealing molecule have been studied subject to the ring current effect thus obtained. This spatial magnetic response property of TSNMRSs dominates the different magnitude of 1H and 13C chemical shifts, especially in the cross-over section of infinitene, which is unequivocally classified as an aromatic molecule based on the deshielding belt of its ring current effect. Differences in aromaticity of infinitene compared with isolated benzene can also be qualified and quantified on the magnetic criterion.

33S NMR: Recent Advances and Applications.

The purpose of this review is to present advances and applications of 33S NMR, which is an underutilized NMR spectroscopy. Experimental NMR aspects in solution, chemical shift tendencies, and quadrupolar relaxation parameters will be briefly summarized. Emphasis will be given to advances and applications in the emerging fields of solid-state and DFT computations of 33S NMR parameters. The majority of the examples were taken from the last twenty years and were selected on the basis of their importance to provide structural, electronic, and dynamic information that is difficult to obtain by other techniques.

Measurements of Nuclear Magnetic Shielding in Molecules.

The origin of nuclear magnetic shielding in diamagnetic molecules is discussed, pointing out various contributions to the shielding from electrons and the effects of intra- and intermolecular interactions. In NMR practice, chemical shifts are determined first as the measure of shielding in observed samples. The descriptions of shielding and chemical shifts are not fully consistent. Gas phase studies permit the withdrawal of intermolecular contributions from shielding and obtaining the magnetic shielding data in isolated molecules. The shielding determination in molecules is possible using at least three methods delivering the reference shielding standards for selected nuclei. The known shielding of one magnetic nucleus can be transferred to other nuclei if the appropriate nuclear magnetic moments are available with satisfactory accuracy. It is possible to determine the nuclear magnetic dipole moments using the most advanced ab initio shielding calculations jointly with the NMR frequencies measurements for small-sized isolated molecules. Helium-3 gas is postulated as all the molecules' primary and universal reference standard of shielding. It can be easily applied using common deuterium lock solvents as the secondary reference standards. The measurements of absolute shielding are available for everyone with the use of standard NMR spectrometers.

The Effect of Hydrogenation on the Contest between Aromaticity and Antiaromaticity in Norcorrole.

Magnetic shielding studies demonstrate that successive hydrogenation of NiII norcorrole (NiNc), a stable molecule combining aromatic and antiaromatic features, first weakens and then eliminates the central antiaromatic region, even though the NiNc antiaromatic "core", a 14-membered conjugated cycle with 16 π electrons, is formally preserved throughout the H2 NiNc-H8 NiNc series. The differences between aromatic and non-aromatic isotropic shielding distributions and nucleus-independent chemical shift (NICS) values in these hydrogenated porphyrin analogues are highlighted by comparing the results for the members of the H2 NiNc-H8 NiNc series to those for the aromatic NiII porphyrin complex. The results strongly support the unexpected and counterintuitive conclusion that H8 NiNc will be nonaromatic, without even a trace of antiaromaticity. Based on these findings, H8 NiNc is predicted to be the most stable member of the H2 NiNc-H8 NiNc series.

Solid-State NMR Spectroscopy to Probe State and Phase Transitions in Frozen Solutions.

Freezing is commonly encountered during the processing and storage of biomacromolecule products. Therefore, understanding the phase and state transitions in pharmaceutical frozen solutions is crucial for the rational development of biopharmaceuticals. Solid-state nuclear magnetic resonance spectroscopy (ssNMR) was used to analyze solutions containing sodium phosphate buffer, histidine, and trehalose. Upon freezing, crystallization of disodium phosphate hydrogen dodecahydrate (Na2HPO4·12H2O, DPDH) and histidine was identified using 31P and 13C ssNMR, respectively, and confirmed by synchrotron X-ray diffractometry (SXRD). Using histidine as a molecular probe and based on the chemical shifts of atoms of interest, the pH of the freeze concentrate was measured. The unfrozen water content in freeze concentrates was quantified by 1H single pulse experiments. 13C-insensitive nuclei enhancement by polarization transfer (INEPT) and cross-polarization (CP) experiments were used as orthogonal tools to characterize the solutes in a "mobile" and a more "solid-like" state in the freeze-concentrated solutions, respectively. The above analyses were applied to a commercial monoclonal antibody (mAb) formulation of dupilumab. This work further establishes ssNMR spectroscopy as a highly capable biophysical tool to investigate the attributes of biopharmaceuticals and thereby provide insights into process optimization and formulation development.

A combined solid-state NMR and quantum chemical calculation study of hydrogen bonding in two forms of α-d-glucose.

Hydrogen bonding plays an important role in the structure and function of a wide range of materials. Solid-state 1H nuclear magnetic resonance (NMR) spectroscopy provides a very sensitive tool to investigate the local structure of hydrogen atoms involved in hydrogen bonding. While there is extensive 1H solid-state NMR data on O-H - - O hydrogen bonding in solid carboxylic acids, there has been no systematic 1H solid-state NMR studies of hydroxyl groups in carbohydrates (and hydroxyl groups in general). With a view to studying the hydrogen bonding in more complex materials such as cellulose polymorphs, we carried out a detailed solid-state 1H NMR investigation of the model compounds α-d-glucose and α-d-glucose monohydrate. Through a combination of fast magic-angle spinning (MAS), combined rotation and multiple pulse spectroscopy (CRAMPS), and two-dimensional (2D) correlation experiments carried out at ultrahigh magnetic fields, it was possible to assign all of the aliphatic (CH), hydroxyl (OH), and water (H2O) 1H chemical shifts in both forms of α-d-glucose. Plane-wave DFT calculations were employed to improve the hydrogen atom positions for α-d-glucose monohydrate and to calculate 1H chemical shifts, providing additional support for the experimentally determined peak assignments. Finally, the relationship between the hydroxyl 1H chemical shifts and their hydrogen bonding geometry was investigated and compared to the well-established relationship for carboxylic acid protons.

Tautomerism of pyridinylbutane-1,3-diones: An NMR and DFT study.

The three possible 1-(n-pyridinyl)butane-1,3-diones (nPM) have been synthesized. Structures, tautomerism, and conformations are investigated by means of DFT calculations. 1 H and 13 C NMR spectra are assigned, and deuterium isotope effects on 13 C chemical shifts have been measured. Analysis of the isotope effects leads to the equilibrium constants of the keto-enol tautomers. Some interesting differences are seen between the three compounds and the phenyl analogs. The isotope effects can also rank the hydrogen bonds of the compounds, with the one with nitrogen in the three positions of the pyridine ring as the weakest. Structures, conformers, energies, and NMR nuclear shieldings are calculated using DFT calculations at the B3LYP/6-311++G(d,p) level.

Specific Proton-Donor Properties of Glycine Betaine. Metric Parameters and Enthalpy of Noncovalent Interactions in its Dimer, Water Complexes and Crystalline Hydrate.

Trimethylglycine (glycine betaine, GB) is an important organic osmolyte that accumulates in various plant species in response to environmental stresses and has significant potential as a bioactive agent with low environmental impact. It is assumed that the hydration of GB is playing an important role in the protective mechanism. The hydration and aggregation properties of GB have not yet been studied in detail at the atomistic level. In this work, noncovalent interactions in the GB dimer and its complexes with water and crystalline monohydrate are studied. Depending on the object, periodic and non-periodic DFT calculations are used. Particular attention is paid to the metric parameters and enthalpies of intermolecular hydrogen bonds. The identification of noncovalent interactions is carried out by means of the Bader analysis of periodic or non-periodic electron density. The enthalpy of hydrogen bonds is estimated using the Rosenberg formula (PCCP 2 (2000) 2699). The specific proton donor properties of glycine betaine are due to its ability to form intermolecular C-H∙∙∙O bonds with the oxygen atom of a water molecule or the carboxylate group of a neighboring GB. The enthalpy of these bonds can be significantly greater than 10 kJ/mol. The water molecule that forms a hydrogen bond with the carboxylate group of GB also interacts with its CH groups through lone pairs of electrons. The C-H∙∙∙O bonds contribute up to 40% of the total entropy of the GB-water interaction, which is about 45 kJ/mol. The possibility of identifying C-H∙∙∙O bonds by the proton nuclear magnetic resonance method is discussed.

Recent Advances in NMR Protein Structure Prediction with ROSETTA.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful method for studying the structure and dynamics of proteins in their native state. For high-resolution NMR structure determination, the collection of a rich restraint dataset is necessary. This can be difficult to achieve for proteins with high molecular weight or a complex architecture. Computational modeling techniques can complement sparse NMR datasets (<1 restraint per residue) with additional structural information to elucidate protein structures in these difficult cases. The Rosetta software for protein structure modeling and design is used by structural biologists for structure determination tasks in which limited experimental data is available. This review gives an overview of the computational protocols available in the Rosetta framework for modeling protein structures from NMR data. We explain the computational algorithms used for the integration of different NMR data types in Rosetta. We also highlight new developments, including modeling tools for data from paramagnetic NMR and hydrogen-deuterium exchange, as well as chemical shifts in CS-Rosetta. Furthermore, strategies are discussed to complement and improve structure predictions made by the current state-of-the-art AlphaFold2 program using NMR-guided Rosetta modeling.

Aromatic Character and Relative Stability of Pyrazoloporphyrin Tautomers and Related Protonated Species: Insights into How Pyrazole Changes the Properties of Carbaporphyrinoid Systems.

Pyrazoloporphyrins (PzPs), which are porphyrin analogues incorporating a pyrazole subunit, are examples of carbaporphyrin-type structures with a carbon atom within the macrocyclic cavity. DFT calculations were used to assess a series of 17 PzP tautomers, nine monoprotonated species and four related diprotonated PzP dications. The geometries of the structures were optimized using M06-2X/6-311++G(d,p), and the relative stabilities computed with the cc-PVTZ functional. Nucleus independent chemical shifts, both NICS(0) and NICS(1)zz, were calculated, and the anisotropy of the induced current density (AICD) plots were generated for all of the species under investigation. The results for free base PzPs show that fully aromatic PzP tautomers are not significantly more stable than weakly aromatic cross-conjugated species. In addition, strongly aromatic structures with internal CH2's are much less stable, a feature that is also seen for protonated PzPs. The degree of planarity for the individual macrocycles does not significantly correlate with the stability of these structures. The results allow significant aromatic conjugation pathways to be identified in many cases, and provide insights into the aromatic properties of this poorly studied system. These investigations also complement experimental results for PzPs and emphasize the need for further studies in this area.

On the Use of Deuterated Organic Solvents without TMS to Report 1H/13C NMR Spectral Data of Organic Compounds: Current State of the Method, Its Pitfalls and Benefits, and Related Issues.

The quite popular, simple but imperfect method of referencing NMR spectra to residual 1H and 13C signals of TMS-free deuterated organic solvents (referred to as Method A) is critically discussed for six commonly used NMR solvents with respect to their δH and δC values that exist in the literature. Taking into account the most reliable data, it was possible to recommend 'best' δX values for such secondary internal standards. The position of these reference points on the δ scale strongly depends on the concentration and type of analyte under study and the solvent medium used. For some solvents, chemically induced shifts (CISs) of residual 1H lines were considered, also taking into account the formation of 1:1 molecular complexes (for CDCl3). Typical potential errors that can occur as a result of improper application of Method A are considered in detail. An overview of all found δX values adopted by users of this method revealed a discrepancy of up to 1.9 ppm in δC reported for CDCl3, most likely caused by the CIS mentioned above. The drawbacks of Method A are discussed in relation to the classical use of an internal standard (Method B), two 'instrumental' schemes in which Method A is often implicitly applied, that is, the default Method C using 2H lock frequencies and Method D based on Ξ values, recommended by the IUPAC but only occasionally used for 1H/13C spectra, and external referencing (Method E). Analysis of current needs and opportunities for NMR spectrometers led to the conclusion that, for the most accurate application of Method A, it is necessary to (a) use dilute solutions in a single NMR solvent and (b) to report δX data applied for the reference 1H/13C signals to the nearest 0.001/0.01 ppm to ensure the precise characterization of new synthesized or isolated organic systems, especially those with complex or unexpected structures. However, the use of TMS in Method B is strongly recommended in all such cases.

An inexpensive density functional theory-based protocol to predict accurate 19 F-NMR chemical shifts.

Thanks to its advantages, 19 F-NMR is an increasingly popular technique for the structural characterization of F-containing molecules, among which polymers, materials, fluorophores, pharmaceuticals, and so forth. However, the computational calculation of the 19 F-NMR chemical shifts, both for prediction and interpretation of experimental spectra, remains a challenge. In this work a density functional theory (DFT) based protocol for the calculation of the chemical shifts is established within the framework of the gauge-independent atomic orbital method, upon verifying the performance of Hartree-Fock and 60 DFT functionals coupled with seven different basis sets. The benchmark is conducted using two sets of molecules, namely one used for testing methods and another used for probing; the former set consists of 134 molecules, the latter 50, yet both of them with F in different chemical environments. Following Bally-Rablen-Tantillo strategy, the scaling parameters and other statistical quantities were computed for each method upon least squares linear regression between experimental and computed chemical shifts. The designed computational workflow is computationally inexpensive and represents a significant improvement with respect to the current state of the art.

Chemical shifts-based similarity restraints improve accuracy of RNA structures determined via NMR.

Determination of structure of RNA via NMR is complicated in large part by the lack of a precise parameterization linking the observed chemical shifts to the underlying geometric parameters. In contrast to proteins, where numerous high-resolution crystal structures serve as coordinate templates for this mapping, such models are rarely available for smaller oligonucleotides accessible via NMR, or they exhibit crystal packing and counter-ion binding artifacts that prevent their use for the chemical shifts analysis. On the other hand, NMR-determined structures of RNA often are not solved at the density of restraints required to precisely define the variable degrees of freedom. In this study we sidestep the problems of direct parameterization of the RNA chemical shifts/structure relationship and examine the effects of imposing local fragmental coordinate similarity restraints based on similarities of the experimental secondary ribose 13C/1H chemical shifts instead. The effect of such chemical shift similarity (CSS) restraints on the structural accuracy is assessed via residual dipolar coupling (RDC)-based cross-validation. Improvements in the coordinate accuracy are observed for all of the six RNA constructs considered here as test cases, which argues for routine inclusion of these terms during NMR-based oligonucleotide structure determination. Such accuracy improvements are expected to facilitate derivation of the chemical shift/structure relationships for RNA.

Prediction of 15 N chemical shifts by machine learning.

We demonstrate the potential for machine learning systems to predict three-dimensional (3D)-relevant NMR properties beyond traditional 1 H- and 13 C-based data, with comparable accuracy to density functional theory (DFT) (but orders of magnitude faster). Predictions of DFT-calculated 15 N chemical shifts for 3D molecular structures can be achieved using a machine learning system-IMPRESSION (Intelligent Machine PREdiction of Shift and Scalar information Of Nuclei), with an accuracy of 6.12-ppm mean absolute error (∼1% of the δ15 N chemical shift range) and an error of less than 20 ppm for 95% of the chemical shifts. It provides less accurate raw predictions of experimental chemical shifts, due to the limited size and chemical space diversity of the training dataset used in its creation, coupled with the limitations of the underlying DFT methodology in reproducing experiment.