NMR Chemical Shift Data Research Articles

Comparison of Antiaromatic Properties in a Series of Structurally Isomeric Naphthothiophene-fused s-Indacenes.

Fusion of aromatic subunits to stabilize an antiaromatic core allows the isolation and study of otherwise unstable paratropic systems. A complete study of a series of six naphthothiophene-fused s-indacene isomers is herein described. Additionally, the structural modifications resulted in increased π-π overlap in the solid state, which was further explored through changing the sterically blocking mesityl group to (triisopropylsilyl)ethynyl in three derivatives. The computed antiaromaticity of the six isomers is compared to the observed physical properties, such as NMR chemical shift, UV-vis, and CV data. We find that the calculations predict the most antiaromatic isomer and give a general estimation of the relative degree of paratropicity for the remaining isomers, when compared to the experimental results.

Structure Determination of Challenging Protein-Peptide Complexes Combining NMR Chemical Shift Data and Molecular Dynamics Simulations.

Intrinsically disordered regions of proteins often mediate important protein-protein interactions. However, the folding-upon-binding nature of many polypeptide-protein interactions limits the ability of modeling tools to predict the three-dimensional structures of such complexes. To address this problem, we have taken a tandem approach combining NMR chemical shift data and molecular simulations to determine the structures of peptide-protein complexes. Here, we use the MELD (Modeling Employing Limited Data) technique applied to polypeptide complexes formed with the extraterminal domain (ET) of bromo and extraterminal domain (BET) proteins, which exhibit a high degree of binding plasticity. This system is particularly challenging as the binding process includes allosteric changes across the ET receptor upon binding, and the polypeptide binding partners can adopt different conformations (e.g., helices and hairpins) in the complex. In a blind study, the new approach successfully modeled bound-state conformations and binding poses, using only protein receptor backbone chemical shift data, in excellent agreement with experimentally determined structures for moderately tight (Kd ∼100 nM) binders. The hybrid MELD + NMR approach required additional peptide ligand chemical shift data for weaker (Kd ∼250 μM) peptide binding partners. AlphaFold also successfully predicts the structures of some of these peptide-protein complexes. However, whereas AlphaFold can provide qualitative peptide rankings, MELD can directly estimate relative binding affinities. The hybrid MELD + NMR approach offers a powerful new tool for structural analysis of protein-polypeptide complexes involving disorder-to-order transitions upon complex formation, which are not successfully modeled with most other complex prediction methods, providing both the 3D structures of peptide-protein complexes and their relative binding affinities.

Low information NMR data guided protein-peptide complex structure determination with MELDXMD.

About 40% of the protein-protein interactions are mediated through small peptide epitope. Knowledge of how a peptide or an intrinsically disordered protein fragment interact with a protein would widen of the understanding of gene regulation, transcription and finally drug discovery. However, the folding upon binding nature of majority of the peptide interacting partner, limits the ability of modeling tools to predict structures of such complexes. To address this problem, we show an integrative approach combining cost-effective NMR chemical shift data and physics based molecular simulations to determine structures of protein-peptide complexes. Here in this presentation, we demonstrate our approach for polypeptide complexes formed with the extraterminal (ET) domain of bromo and extraterminal domain (BET) proteins, which exhibit a high degree of binding plasticity. This system is particularly challenging as the binding process includes allosteric changes across the ET receptor upon binding, and the polypeptide binding partners can form different conformations (e.g., helices and hairpins) in the complex. In a blind study, the new approach successfully predicts bound-state structure, using only backbone chemical shift data, in excellent agreement with experimental structure. We show, this approach is successful on not only strongly interacting complexes, but also in the case of weak binder, this method has been successful. MELD+NMR also predicts relative binding affinities of different peptides, consistent with the experimental values. The hybrid MELD+NMR approach provides a powerful new tool for structural analysis of protein-polypeptide complexes involving disorder to order transitions upon complex formation which are not successfully modeled with most other complex predication methods including state of the art docking methods, providing 3D atomistic structures of complexes as well as their relative binding affinities.

Structure of silk I (Bombyx mori silk fibroin before spinning) in the dry and hydrated states studied using 13C solid-state NMR spectroscopy

Nowadays, much attention has been paid to Bombyx mori silk fibroin (SF) by many researchers because of excellent physical properties and biocompatibility. These superior properties originate from the structure of SF and therefore, the structural analysis is a key to clarify the superiority. Here we concentrated on silk I structure (SF structure before spinning). We showed that silk I* (the structure of (GAGAGS)n which is a main part of SF) is a repeated type II β-turn, neither α-helix nor random coil, from the conformation-dependent 13C NMR chemical shift data. This conclusion is different from that obtained using IR by many researchers. Next, the formation of silk I* structure was investigated at molecular level using 13C solid-state NMR spectroscopy. Three kinds of 13C INEPT, CP/MAS and DD/MAS NMR spectra were observed for SF, [3-13C]Ser- and [3-13C]Tyr-SF, the crystalline fraction obtained by chymotrypsin treatment of SF and their model peptide with silk I structures in the dry and hydrated states. Especially, the presence of the sequences containing Tyr, (((GX)m1GY)m2 where X = A or V) with random coil conformations adjacent to (GAGAGS)n is an essence to get water-soluble SF and the formation of silk I* structure of (GAGAGS)n.

Complete 1H and 13C NMR chemical shift assignments of mono-to tetrasaccharides as basis for NMR chemical shift predictions of oligo- and polysaccharides using the computer program CASPER

Carbohydrate structure can be elucidated or confirmed by using NMR spectroscopy as the prime technique. Prediction of 1H and 13C NMR chemical shifts by computational approaches makes this assignment process more efficient and the program CASPER can perform this task rapidly. It does so by relying on chemical shift data of mono-, di-, and trisaccharides. In order to improve accuracy and quality of these predictions we have assigned 1H and 13C NMR chemical shifts of 30 monosaccharides, 17 disaccharides, 10 trisaccharides and one tetrasaccharide; in total 58 compounds. Due to different rotamers, ring forms, α- and β-anomeric forms and pD conditions this resulted in 74 1H and 13C NMR chemical shift data sets, all of which were refined using total line-shape analysis for the 1H resonances in order to obtain accurate chemical shifts. Subsequent NMR chemical shift predictions for three sialic acid-containing oligosaccharides, viz., GD1a, a disialyl-LNnT hexasaccharide and a polysialic acid-lactose decasaccharide, and NMR-based structural elucidations of two O-antigen polysaccharides from E. coli O174 were performed by the CASPER program (http://www.casper.organ.su.se/casper/) resulting in very good to excellent agreement between experimental and predicted data thereby demonstrating its utility for carbohydrate compounds that have been chemically or enzymatically synthesized, structurally modified or isolated from nature.

Molecular Basis of Small-Molecule Binding to α-Synuclein.

Intrinsically disordered proteins (IDPs) are implicated in many human diseases. They have generally not been amenable to conventional structure-based drug design, however, because their intrinsic conformational variability has precluded an atomic-level understanding of their binding to small molecules. Here we present long-time-scale, atomic-level molecular dynamics (MD) simulations of monomeric α-synuclein (an IDP whose aggregation is associated with Parkinson’s disease) binding the small-molecule drug fasudil in which the observed protein–ligand interactions were found to be in good agreement with previously reported NMR chemical shift data. In our simulations, fasudil, when bound, favored certain charge–charge and π-stacking interactions near the C terminus of α-synuclein but tended not to form these interactions simultaneously, rather breaking one of these interactions and forming another nearby (a mechanism we term dynamic shuttling). Further simulations with small molecules chosen to modify these interactions yielded binding affinities and key structural features of binding consistent with subsequent NMR experiments, suggesting the potential for MD-based strategies to facilitate the rational design of small molecules that bind with disordered proteins.

SWCNTs Interaction with Dopamine and Serotonin Anticancer Through QM/MM Methods: A Drug Delivery Approaches

Objetive: Dopamine and Serotonin are the two important biological transmitters that have hormonal activities and responsible for happiness and felling well. The aim of this article was to study theoretically the structure features of Dopamine and Serotonin in the complex of single-walled carbon nanotube as a neurotransmitter. Material and Methods: The structure of Dopamine and Serotonin binding with SWCNT with four different diameters (7.0,7.5,7.7,10.0 nm) was studied by using molecular mechanic (MM) and quantum mechanic (QM). The remarkable energies including potential energy, total energy and kinetic energy in time of simulation 10 ns steps in two temperatures (298, 310 kelvin degree) were investigated by Monte Carlo method with opls force filed. NMR shielding tensor data by B3LYP level of theory with 6-31 G(d) as a basis set and semi empirical method have been also fulfilled. Results: Theoretical computations were performed to study NMR chemical shift data including magnetic shielding tensor (σ, ppm), shielding asymmetry (η), magnetic shielding anisotropy (σaniso), magnetic shielding isotropy (σiso) , skew of a tensor (Κ) and chemical shift anisotropy (Δσ) and span (Ω) at various rotation angles around a specific rotation, physical and chemical properties of atomic nuclei. Semi empirical calculations such as total energy, binding energy, isolated atomic energy, electronic energy, core–core interaction and heat of formation in AM1 were revealed. Conclusion: It is figured out in Monte Carlo method our two specific drug and its nanotube with small diameter are the most stable one than the others. The larger diameter leads the combination stability into lower value.

Investigation of different SWCNTs interaction with dopamine and serotonin anticancers: a theoretical study

Carbon nano tubes (CNTS) have two basic structure as single-walled and multi-walled based on hexagonal plexus of carbon atoms. CNTs can serve as platforms to conjugate other compounds specially in medications purposes by immobilization of biomolecules at their surface. Dopamine and serotonin are two biological molecules which have bifunctional activities as hormone and neurotransmitter. These two molecules have important roles as neurotransmitters in the central and peripheral nervous systems but serotonin functions as a mood regulator, while dopamine is connected to the “pleasure center”. In this article we optimized molecular and structural properties of connected dopamine and serotonin with SWNTS with four different diameters (7.0,7.5,7.7 and 10.0 nm) by using molecular quantum methods such as NMR shielding tensor data by B3LYP level of theory with 6-31 G(d) as a basis set, mk and frequency methods. Theoretical computations were performed to study NMR chemical shift data including magnetic shielding tensor (σ, ppm), shielding asymmetry (η), magnetic shielding anisotropy (σaniso), magnetic shielding isotropy (σiso) , skew of a tensor (Κ) and chemical shift anisotropy (Δσ) and span (Ω) at various rotation angles around a specific rotation , physical and chemical properties of atomic nuclei , frequency data by B3LYP/6-31g level of theory and POP method using gaussian 09 program.

ODiNPred: comprehensive prediction of protein order and disorder

Structural disorder is widespread in eukaryotic proteins and is vital for their function in diverse biological processes. It is therefore highly desirable to be able to predict the degree of order and disorder from amino acid sequence. It is, however, notoriously difficult to predict the degree of local flexibility within structured domains and the presence and nuances of localized rigidity within intrinsically disordered regions. To identify such instances, we used the CheZOD database, which encompasses accurate, balanced, and continuous-valued quantification of protein (dis)order at amino acid resolution based on NMR chemical shifts. To computationally forecast the spectrum of protein disorder in the most comprehensive manner possible, we constructed the sequence-based protein order/disorder predictor ODiNPred, trained on an expanded version of CheZOD. ODiNPred applies a deep neural network comprising 157 unique sequence features to 1325 protein sequences together with the experimental NMR chemical shift data. Cross-validation for 117 protein sequences shows that ODiNPred better predicts the continuous variation in order along the protein sequence, suggesting that contemporary predictors are limited by the quality of training data. The inclusion of evolutionary features reduces the performance gap between ODiNPred and its peers, but analysis shows that it retains greater accuracy for the more challenging prediction of intermediate disorder.

Backbone and sidechain NMR assignments for the ribosome maturation factor RimP from Escherichia coli.

Ribosome biogenesis is an energetically expensive and complex cellular process that involves the coordinated folding of the ribosomal RNA and dozens of ribosomal proteins. It proceeds along multiple parallel pathways and is guided by trans-acting factors called ribosome assembly factors. Although this process has been studied for decades, there are still many open questions regarding the role of the ribosome assembly factors in directing the folding of ribosome biogenesis intermediates. RimP is one of the early acting factors and guides the assembly of the small 30S ribosomal subunit by facilitating the binding of ribosomal proteins uS5 and uS12. Here we report the virtually complete 1H, 15N, and 13C chemical shift assignment of RimP from Escherichia coli. The NMR chemical shift data, deposited in the BMRB data bank under Accession No. 28014, indicates a widely folded protein composed of three alpha helices and eight beta strands.

Structural and energetic investigation on the host/guest inclusion process of benzyl isothiocyanate into β-cyclodextrin using dispersion-corrected DFT calculations

The formation of host-guest complex between benzyl isothiocyanate (BITC) and β-cyclodextrin (β-CD) was studied using dispersion-corrected density functional theory calculations. The complexation process was monitored using molecular docking simulations, natural bond orbital (NBO) technique, nuclear magnetic resonance (1H NMR) chemical shift calculations and non-covalent interactions (NCI) analysis. All these approaches are consistent with experimental findings. The calculated complexation energy was negative indicating the formation of inclusion complex. The most stable complexation of BITC involves the inclusion of its aromatic moiety in β-CD cavity (model A) in accord with experimental NMR chemical shift data. The non-covalent interactions (NCI) based on the reduced density gradient (RDG) analysis reveal that mainly weak Van der Waals intermolecular interactions between BITC and β-CD provide and ensure stability for the complexation process.

1H, 13C and 15N resonance assignments of translation initiation factor 3 from Pseudomonas aeruginosa.

Translation initiation factor 3 (IF3) is one of the three protein factors that bind to the small ribosomal subunit and it is required for the initiation of protein biosynthesis in bacteria. IF3 contains two independent domains, N- and C-terminal domains, which are connected by a lysine-rich interdomain linker. IF3 undergoes large-scale movements and conformational changes upon binding to the 30S subunit and also during the functional regulation of initiation. However, the precise dynamic interplay of the two domains and the molecular mechanism of IF3 is not well understood. A high-resolution 3D structure of a complete IF3 in bacteria has not been solved. Pseudomonas aeruginosa, a gram-negative opportunistic pathogen, is a primary cause of nosocomial infections in humans. Here we report the NMR chemical shift assignments of IF3 from P. aeruginosa as the first step toward NMR structure determination and interaction studies. Secondary structure analyses deduced from the NMR chemical shift data identified nine β-strands and four α-helices arranged in the sequential order β1-β2-α1-β3-β4-α2-β5-α3-β6-α4-β7-β8-β9.

Deconvolution of fast exchange equilibrium states in NMR spectroscopy using virtual reference standards and probability theory.

A methodology for deconvolution of fast exchange equilibrium states in NMR spectroscopy (DFEQNMR) was developed based on DFT-GIAO NMR chemical shift prediction and a probability theory algorithm. Proof-of-concept studies were performed to estimate the protonation state of N-containing organic molecules involving fast proton exchange equilibrium and evaluate the solution tautomerism of a purine derivative. DFT-GIAO calculations were optimized to achieve good accuracy in 13C, 1H and 15N chemical shift prediction for protonated species. The probability theory algorithm enabled the determination of solution species ratios and yielded 95% confidence regions by comparing experimental and simulated chemical shift data sets. The calculation showed good accuracy for model partial salts with various functionalities and application in structure elucidation of complex natural product partial salts was also demonstrated. This method showed promising potential in acquisition of important insight into fast exchange equilibrium systems with only one experimental NMR chemical shift data set.

Using NMR Chemical Shifts and Cryo-EM Density Restraints in Iterative Rosetta-MD Protein Structure Refinement.

Cryo-EM has become one of the prime methods for protein structure elucidation, frequently yielding density maps with near-atomic or medium resolution. If protein structures cannot be deduced unambiguously from the density maps, computational structure refinement tools are needed to generate protein structural models. We have previously developed an iterative Rosetta-MDFF protocol that used cryo-EM densities to refine protein structures. Here we show that, in addition to cryo-EM densities, incorporation of other experimental restraints into the Rosetta-MDFF protocol further improved refined structures. We used NMR chemical shift (CS) data integrated with cryo-EM densities in our hybrid protocol in both the Rosetta step and the molecular dynamics (MD) simulations step. In 15 out of 18 cases for all MD rounds, the refinement results obtained when density maps and NMR chemical shift data were used in combination outperformed those of density map-only refinement. Notably, the improvement in refinement was highest when medium and low-resolution density maps were used. With our hybrid method, the RMSDs of final models obtained were always better than the RMSDs obtained by our previous protocol with just density refinement for both medium (6.9 Å) and low (9 Å) resolution maps. For all the six test proteins with medium resolution density maps (6.9 Å), the final refined structure RMSDs were lower for the hybrid method than for the cryo-EM only refinement. The final refined RMSDs were less than 1.5 Å when our hybrid protocol was used with 4 Å density maps. For four out of the six proteins the final RMSDs were even less than 1 Å. This study demonstrates that by using a combination of cryo-EM and NMR restraints, it is possible to refine structures to atomic resolution, outperforming single restraint refinement. This hybrid protocol will be a valuable tool when only low-resolution cryo-EM density data and NMR chemical shift data are available to refine structures.

Bayesian-Maximum-Entropy Reweighting of IDP Ensembles Based on NMR Chemical Shifts

Bayesian and Maximum Entropy approaches allow for a statistically sound and systematic fitting of experimental and computational data. Unfortunately, assessing the relative confidence in these two types of data remains difficult as several steps add unknown error. Here we propose the use of a validation-set method to determine the balance, and thus the amount of fitting. We apply the method to synthetic NMR chemical shift data of an intrinsically disordered protein. We show that the method gives consistent results even when other methods to assess the amount of fitting cannot be applied. Finally, we also describe how the errors in the chemical shift predictor can lead to an incorrect fitting and how using secondary chemical shifts could alleviate this problem.

Spectroscopic (FT-IR, FT-Raman, NMR and UV-Vis), ELF, LOL, NBO, and Fukui function investigations on (5-bromo-benzofuran-3-yl)-acetic acid hydrazide (5BBAH): Experimental and theoretical approach

Experimental and theoretical spectroscopic studies were performed for the (5-Bromo-benzofuran-3-yl)-acetic acid hydrazide (5BBAH) molecule. The research work was performed in two levels. In the first level, experimental FT-IR, FT-Raman, NMR (1H, 13C) chemical shift and UV-Vis spectral data were recorded. In the next level the theoretical computations have been conducted from DFT/B3LYP/6-311++G (d, p) basis level. Initially, the theoretical geometrical parameters were obtained and matched with related experimental parameters. The computed FT-IR and FT-Raman frequencies were collected from same basis level and matched with experimental data. The vibrational assignments were achieved on the PED of individual vibrational modes. The theoretical NMR chemical shifts were compared with recorded experimental data. Theoretical (TD-DFT) and experimental UV-Vis absorption wavelengths (λ) were compared with each other. In addition, FMO, NBO, MEP, ELF, LOL and Fukui function analysis were performed.

Guaianolides from Ormenis mixta: Structural Insights and Evaluation of Their Anti-inflammatory Profile.

In this paper, the isolation of five new guaianolides (1: - 5: ) and four (6: - 9: ) known sesquiterpenes from Ormenis mixta aerial parts is reported. The structural determination of the guaianolides was obtained by NMR spectroscopic data, as well as MS experiments. Their relative configurations were assigned by a combined quantum mechanical/NMR approach, comparing the experimental 13C/1H NMR chemical shift data and 1 J H-H homonuclear coupling constants with the related predicted values. The isolates were assayed for their anti-inflammatory potential evaluating nitric oxide release and cyclooxygenase-2 expression in J774A.1 macrophages treated with lipopolysaccharide from Escherichia coli. Our results indicated that, among the tested compounds, 1: - 3: , and 7: were able to inhibit nitric oxide release, while all were able to inhibit cyclooxygenase-2 expression with different potencies.

Quality and bias of protein disorder predictors

Disorder in proteins is vital for biological function, yet it is challenging to characterize. Therefore, methods for predicting protein disorder from sequence are fundamental. Currently, predictors are trained and evaluated using data from X-ray structures or from various biochemical or spectroscopic data. However, the prediction accuracy of disordered predictors is not calibrated, nor is it established whether predictors are intrinsically biased towards one of the extremes of the order-disorder axis. We therefore generated and validated a comprehensive experimental benchmarking set of site-specific and continuous disorder, using deposited NMR chemical shift data. This novel experimental data collection is fully appropriate and represents the full spectrum of disorder. We subsequently analyzed the performance of 26 widely-used disorder prediction methods and found that these vary noticeably. At the same time, a distinct bias for over-predicting order was identified for some algorithms. Our analysis has important implications for the validity and the interpretation of protein disorder, as utilized, for example, in assessing the content of disorder in proteomes.

Two non-identical twins in one unit cell: characterization of 34π aromatic core-modified octaphyrins, their structural isomers and anion bound complexes.

Four different core-modified planar 34π octaphyrins (10, 11, 13, and 15) which exhibit rotational isomerism have been synthesized and characterized both in solution and solid states. Octaphyrins 10, 11 and 13 show two inseparable isomers A and B which crystallize in the same unit cell. However, 15 forms two identical isomers of A. Structurally, the two isomers in 10, 11 and 13 (A and B) are different only in the ring inversion of one of the thiophene or selenophene rings present in the terthiophene subunit of the macrocycle. In isomer A, the middle thiophene or selenophene rings are inverted, while in isomer B, the terminal thiophene rings are inverted. The 1H NMR spectrum of these macrocycles shows peaks assignable to protons of both the isomers in toluene D8. The single crystal structure analysis of 10 reveals the presence of both isomers 10A and 10B in a single unit cell with the P21/n space group. Both the isomers exhibit aromatic behaviour in the freebase form. Protonation of pyrrole nitrogens leads to exclusive formation of isomer B for 10 and 11. However, both the isomers are present upon protonation of 13 where the central heterocyclic ring of terthiophene subunits has thiophene and selenophene rings. Octaphyrin 15 crystallizes in the P21/c space group and exclusively isomer A was formed in the reaction. Protonation of pyrrole nitrogens leads to significant increases in aromaticity as revealed by 1H NMR chemical shift data. The NICS values calculated for the individual heterocyclic rings before and after protonation support such a conclusion. The AICD plots exhibit clockwise orientation of current density vectors suggesting the presence of diatropic ring current in the octaphyrins. Energy calculations at the M06L/CC-pVTZ//M06L/6-31G** level qualitatively account for exclusive stabilization of a particular isomer relative to the other upon protonation. To the best of our knowledge 10 represents the first example in expanded porphyrin chemistry where two different structural isomers crystallize in a single unit cell.

Nonequivalence of Second Sphere "Noncatalytic" Residues in Pentaerythritol Tetranitrate Reductase in Relation to Local Dynamics Linked to H-Transfer in Reactions with NADH and NADPH Coenzymes.

Many enzymes that catalyze hydride transfer reactions work via a mechanism dominated by quantum mechanical tunneling. The involvement of fast vibrational modes of the reactive complex is often inferred in these reactions, as in the case of the NAD(P)H-dependent pentaerythritol tetranitrate reductase (PETNR). Herein, we interrogated the H-transfer mechanism in PETNR by designing conservative (L25I and I107L) and side chain shortening (L25A and I107A) PETNR variants and using a combination of experimental approaches (stopped-flow rapid kinetics, X-ray crystallography, isotope/temperature dependence studies of H-transfer and NMR spectroscopy). X-ray data show subtle changes in the local environment of the targeted side chains but no major structural perturbation caused by mutagenesis of these two second sphere active site residues. However, temperature dependence studies of H-transfer revealed a coenzyme-specific and complex thermodynamic equilibrium between different reactive configurations in PETNR–coenzyme complexes. We find that mutagenesis of these second sphere “noncatalytic” residues affects differently the reactivity of PETNR with NADPH and NADH coenzymes. We attribute this to subtle, dynamic structural changes in the PETNR active site, the effects of which impact differently in the nonequivalent reactive geometries of PETNR−NADH and PETNR−NADPH complexes. This inference is confirmed through changes observed in the NMR chemical shift data for PETNR complexes with unreactive 1,4,5,6-tetrahydro-NAD(P) analogues. We show that H-transfer rates can (to some extent) be buffered through entropy–enthalpy compensation, but that use of integrated experimental tools reveals hidden complexities that implicate a role for dynamics in this relatively simple H-transfer reaction. Similar approaches are likely to be informative in other enzymes to understand the relative importance of (distal) hydrophobic side chains and dynamics in controlling the rates of enzymatic H-transfer.