NMR Structure Determination Research Articles

Synthesis, characterization and computational studies on (E)-4-(1-((2-hydroxyphenyl)imi no) hexyl)-5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one

A coupling precursor, 4-hexanoyl-5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one, 1 synthesized by the reaction of 3-methyl-1-phenyl-1H-pyrazol-5-one with hexanoyl chloride was reacted with 2-aminophenol to synthesize (E)-4-(1-((2-hydroxyphenyl)imino)hexyl)-5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one, 1a. The compounds were characterized by conventional spectroscopic techniques, 1H and 13C NMR, UV–Visible, FTIR spectroscopy and single crystal X-ray crystallograpic structural determinations and computational techniques. To augment the experimental data, DFT calculations were carried. Molecular dockings were used to predict the interactions between, 1a with some biological targets. The binding free energies in the Plasmepsin II active sites indicated anti−malarial activity against P. falciparum in epidermal growth factor receptor (EGFR) inhibition, anti-inflammatory properties against human peroxiredoxin 5, and anticancer properties. With binding energies less than −5.00 kcal/mol, the results indicate that 1a is a potential drug target for anti-inflammatory, anti-malarial and anti-cancer properties.

Discovery of Five Classes of Bacterial Defensins: Ancestral Precursors of Defensins from Eukarya?

Defensins are present in many organisms and are divided into two evolutionary groups, termed cis- and trans-defensins. Cis-defensins have only recently been reported in bacteria, and knowledge of these defensins is limited, with no family classification. Here, we describe the identification of 74 cis-defensins from bacteria and propose five classes for their classification. We also report the first NMR structure determination of a Myxoccocus xanthus defensin, as well as its in silico expression analysis. Xanthusin-1 has a unique structure among the published defensins, which could indicate that the proposed class II peptides constitute a separate group of defensins. Xanthusin-1 gene expression was observed in casitone-based and Streptomyces coelicolor coculture-grown media. Our results demonstrate a wider distribution of defensins outside the Eukarya domain, shedding light on the origin and distribution of defensins. The sharing of three disulfide defensins between bacteria and eukaryotes points to a possible prokaryotic origin of the CSαβ motif. Moreover, the identification of defensins in Gram-positive and Gram-negative bacteria indicates an early origin but with many gene losses during the evolutionary process, similar to findings for eukaryotic defensins.

N-terminal domain of polypyrimidine-tract binding protein is a dynamic folding platform for adaptive RNA recognition.

The N-terminal RNA recognition motif domain (RRM1) of polypyrimidine tract binding protein (PTB) forms an additional C-terminal helix α3, which docks to one edge of the β-sheet upon binding to a stem-loop RNA containing a UCUUU pentaloop. Importantly, α3 does not contact the RNA. The α3 helix therefore represents an allosteric means to regulate the conformation of adjacent domains in PTB upon binding structured RNAs. Here we investigate the process of dynamic adaptation by stem-loop RNA and RRM1 using NMR and MD in order to obtain mechanistic insights on how this allostery is achieved. Relaxation data and NMR structure determination of the free protein show that α3 is partially ordered and interacts with the domain transiently. Stem-loop RNA binding quenches fast time scale dynamics and α3 becomes ordered, however microsecond dynamics at the protein-RNA interface is observed. MD shows how RRM1 binding to the stem-loop RNA is coupled to the stabilization of the C-terminal helix and helps to transduce differences in RNA loop sequence into changes in α3 length and order. IRES assays of full length PTB and a mutant with altered dynamics in the α3 region show that this dynamic allostery influences PTB function in cultured HEK293T cells.

Hidden Structural States of Proteins Revealed by Conformer Selection with AlphaFold-NMR.

Recent advances in molecular modeling using deep learning can revolutionize our understanding of dynamic protein structures. NMR is particularly well-suited for determining dynamic features of biomolecular structures. The conventional process for determining biomolecular structures from experimental NMR data involves its representation as conformation-dependent restraints, followed by generation of structural models guided by these spatial restraints. Here we describe an alternative approach: generating a distribution of realistic protein conformational models using artificial intelligence-(AI-) based methods and then selecting the sets of conformers that best explain the experimental data. We applied this conformational selection approach to redetermine the solution NMR structure of the enzyme Gaussia luciferase. First, we generated a diverse set of conformer models using AlphaFold2 (AF2) with an enhanced sampling protocol. The models that best-fit NOESY and chemical shift data were then selected with a Bayesian scoring metric. The resulting models include features of both the published NMR structure and the standard AF2 model generated without enhanced sampling. This "AlphaFold-NMR" protocol also generated an alternative "open" conformational state that fits nearly as well to the overall NMR data but accounts for some NOESY data that is not consistent with first "closed" conformational state; while other NOESY data consistent with this second state are not consistent with the first conformational state. The structure of this "open" structural state differs from that of the "closed" state primarily by the position of a thumb-shaped loop between α-helices H5 and H6, revealing a cryptic surface pocket. These alternative conformational states of Gluc are supported by "double recall" analysis of NOESY data and AF2 models. Additional structural states are also indicated by backbone chemical shift data indicating partially-disordered conformations for the C-terminal segment. Considered as a multistate ensemble, these multiple states of Gluc together fit the NOESY and chemical shift data better than the "restraint-based" NMR structure and provide novel insights into its structure-dynamic-function relationships. This study demonstrates the potential of AI-based modeling with enhanced sampling to generate conformational ensembles followed by conformer selection with experimental data as an alternative to conventional restraint satisfaction protocols for protein NMR structure determination.

AlphaFold2 as a replacement for solution NMR structure determination of small proteins: Not so fast!

The determination of a protein’s structure is often a first step towards the development of a mechanistic understanding of its function. Considerable advances in computational protein structure prediction have been made in recent years, with AlphaFold2 (AF2) emerging as the primary tool used by researchers for this purpose. While AF2 generally predicts accurate structures of folded proteins, we present here a case where AF2 incorrectly predicts the structure of a small, folded and compact protein with high confidence. This protein, pro-interleukin-18 (pro-IL-18), is the precursor of the cytokine IL-18. Interestingly, the structure of pro-IL-18 predicted by AF2 matches that of the mature cytokine, and not the corresponding experimentally determined structure of the pro-form of the protein. Thus, while computational structure prediction holds immense promise for addressing problems in protein biophysics, there is still a need for experimental structure determination, even in the context of small well-folded, globular proteins.

Efficient Segmental Isotope Labeling of Integral Membrane Proteins for High-Resolution NMR Studies.

High-resolution structural NMR analyses of membrane proteins are challenging due to their large size, resulting in broad resonances and strong signal overlap. Among the isotope labeling methods that can remedy this situation, segmental isotope labeling is a suitable strategy to simplify NMR spectra and retain high-resolution structural information. However, protein ligation within integral membrane proteins is complicated since the hydrophobic protein fragments are insoluble, and the removal of ligation side-products is elaborate. Here, we show that a stabilized split-intein system can be used for rapid and high-yield protein trans-splicing of integral membrane proteins under denaturing conditions. This setup enables segmental isotope labeling experiments within folded protein domains for NMR studies. We show that high-quality NMR spectra of markedly reduced complexity can be obtained in detergent micelles and lipid nanodiscs. Of note, the nanodisc insertion step specifically selects for the ligated and correctly folded membrane protein and simultaneously removes ligation byproducts. Using this tailored workflow, we show that high-resolution NMR structure determination is strongly facilitated with just two segmentally isotope-labeled membrane protein samples. The presented method will be broadly applicable to structural and dynamical investigations of (membrane-) proteins and their complexes by solution and solid-state NMR but also other structural methods where segmental labeling is beneficial.

CuI Promoted Efficient Synthesis and Antimicrobial Activity of Substituted 8,8-Dimethyl-5-phenyl-2-(pyrazin-2-yl)-5,7,8,9-tetrahydro-6H-[1,3,4]thiadiazolo[2,3-b]quinazolin-6-one

In present study, an efficient synthesis of substituted 5,7,8,9-tetrahydro-6H-[1,3,4]thiadiazolo[2,3-b]quinazolin-6-one compounds promoted by CuI as catalyst was carried out. These derivatives were obtained from 1,3,4-thiadiazol-2-amine, dimedone and substituted aromatic aldehyde in presence of CuI in ethanol as solvent at 70 ºC. Initially, 1,3,4-thiadiazol-2-amine was synthesized from pyrazine-3- carboxylic acid reacted with thiosemicarbazide in the presence of 50% H2SO4 in acetonitrile at 70 ºC. All the newly obtained derivatives were evaluated by the spectroscopic techniques such as 1H NMR, 13C NMR and LCMS and structural determination of titled analogous were analyzed by elemental analysis. The antibacterial activities of the newly synthesized compounds were also screened.

Feasibility of Domain Segmentation of B19V VP1u Using Intein Technology for Structural Studies.

Parvovirus B19 (B19V) is a human pathogen, and the minor capsid protein of B19V possesses a unique N terminus called VP1u that plays a crucial role in the life cycle of the virus. The objective of this study was to develop a method for domain segmentation of B19 VP1u using intein technology, particularly its receptor binding domain (RBD) and phospholipase A2 (PLA2) domain. RBD and PLA2 domains of VP1u were each fused to the DnaE split inteins derived from the Nostoc punctiforme. Each of these precursor proteins was expressed in E. coli. Combining the purified precursors in equal molar ratios resulted in the formation of full-length VP1u. Furthermore, Circular Dichroism (CD) spectroscopy and PLA2 assays were used to probe the structure and activity of the newly formed protein. The CD spectrum of the full length VP1u confirmed the secondary structure of protein, while the PLA2 assay indicated minimal disruption in enzymatic activity. This method would allow for the selective incorporation of NMR-active isotopes into either of the VP1u domains, which can reduce signal overlap in NMR structural determination studies.

Improving Geometric Validation Metrics and Ensuring Consistency with Experimental Data through TrioSA: An NMR Refinement Protocol.

Protein model refinement a the crucial step in improving the quality of a predicted protein model. This study presents an NMR refinement protocol called TrioSA (torsion-angle and implicit-solvation-optimized simulated annealing) that improves the accuracy of backbone/side-chain conformations and the overall structural quality of proteins. TrioSA was applied to a subset of 3752 solution NMR protein structures accompanied by experimental NMR data: distance and dihedral angle restraints. We compared the initial NMR structures with the TrioSA-refined structures and found significant improvements in structural quality. In particular, we observed a reduction in both the maximum and number of NOE (nuclear Overhauser effect) violations, indicating better agreement with experimental NMR data. TrioSA improved geometric validation metrics of NMR protein structure, including backbone accuracy and the secondary structure ratio. We evaluated the contribution of each refinement element and found that the torsional angle potential played a significant role in improving the geometric validation metrics. In addition, we investigated protein-ligand docking to determine if TrioSA can improve biological outcomes. TrioSA structures exhibited better binding prediction compared to the initial NMR structures. This study suggests that further development and research in computational refinement methods could improve biomolecular NMR structural determination.

Facilitating the structural characterisation of non-canonical amino acids in biomolecular NMR.

Peptides and proteins containing non-canonical amino acids (ncAAs) are a large and important class of biopolymers. They include non-ribosomally synthesised peptides, post-translationally modified proteins, expressed or synthesised proteins containing unnatural amino acids, and peptides and proteins that are chemically modified. Here, we describe a general procedure for generating atomic descriptions required to incorporate ncAAs within popular NMR structure determination software such as CYANA, CNS, Xplor-NIH and ARIA. This procedure is made publicly available via the existing Automated Topology Builder (ATB) server (https://atb.uq.edu.au, last access: 17 February 2023) with all submitted ncAAs stored in a dedicated database. The described procedure also includes a general method for linking of sidechains of amino acids from CYANA templates. To ensure compatibility with other systems, atom names comply with IUPAC guidelines. In addition to describing the workflow, 3D models of complex natural products generated by CYANA are presented, including vancomycin. In order to demonstrate the manner in which the templates for ncAAs generated by the ATB can be used in practice, we use a combination of CYANA and CNS to solve the structure of a synthetic peptide designed to disrupt Alzheimer-related protein-protein interactions. Automating the generation of structural templates for ncAAs will extend the utility of NMR spectroscopy to studies of more complex biomolecules, with applications in the rapidly growing fields of synthetic biology and chemical biology. The procedures we outline can also be used to standardise the creation of structural templates for any amino acid and thus have the potential to impact structural biology more generally.

Missing Pieces in Structure Puzzles: How Hyperpolarized NMR Spectroscopy Can Complement Structural Biology and Biochemistry.

Structure determination lies at the heart of many biochemical research programs. However, the "giants": X-ray diffraction, electron microscopy, molecular dynamics simulations, and nuclear magnetic resonance, among others, leave quite a few dark spots on the structural pictures drawn of proteins, nucleic acids, membranes, and other biomacromolecules. For example, structural models under physiological conditions or of short-lived intermediates often remain out of reach of the established experimental methods. This account frames the possibility of including hyperpolarized, that is, dramatically signal-enhanced NMR in existing workflows to fill these spots with detailed depictions. We highlight how integrating methods based on dissolution dynamic nuclear polarization can provide valuable complementary information about formerly inaccessible conformational spaces for many systems. A particular focus will be on hyperpolarized buffers to facilitate the NMR structure determination of challenging systems.

Specific Signal Enhancement on an RNA-Protein Interface by Dynamic Nuclear Polarization.

Sensitivity and specificity are both crucial for the efficient solid-state NMR structure determination of large biomolecules. We present an approach that features both advantages by site-specific enhancement of NMR spectroscopic signals from the protein-RNA binding site within a ribonucleoprotein (RNP) by dynamic nuclear polarization (DNP). This approach uses modern biochemical techniques for sparse isotope labeling and exploits the molecular dynamics of 13 C-labeled methyl groups exclusively present in the protein. These dynamics drive heteronuclear cross relaxation and thus allow specific hyperpolarization transfer across the biomolecular complex's interface. For the example of the L7Ae protein in complex with a 26mer guide RNA minimal construct from the box C/D complex in archaea, we demonstrate that a single methyl-nucleotide contact is responsible for most of the polarization transfer to the RNA, and that this specific transfer can be used to boost both NMR spectral sensitivity and specificity by DNP.

Isolation and Reactivity of Stannylenoids Stabilized by Amido/Imino Ligands.

The reaction of the lithium aryl(silyl)amide Dipp(i Pr3 Si)NLi (Dipp=2,6-i Pr2 C6 H3 ) with one equivalent of SnCl2 in THF gave a novel stannylenoid Dipp(i Pr3 Si)NSnCl⋅LiCl(THF)2 . Heating the solution of amidostannylenoid in toluene to 80 °C resulted in dimeric amido(chloro)stannylene [Dipp(i Pr3 Si)NSnCl]2 , which can be converted to bis(amido)stannylene Sn[N(Dipp)(i Pr3 Si)]2 and amido(imino)stannylene Sn[N(Dipp)(i Pr3 Si)][IPrN] (IPrN=bis(2,6-diisopropylphenyl)imidazolin-2-imino). Treatment of bis(imino)stannylenoid [IPrN]2 Sn(Cl)Li with N2 O resulted in the dimeric complex [IPrNSn(Cl)OLi]2 . All compounds were characterized by NMR, elementary analysis, and X-ray structural determination.

Solid-State NMR Structure of Amyloid-β Fibrils.

Amyloid fibrils are involved in a number of diseases and notably play a role in neurodegeneration, where they are present in plaques in the brain. Their structure determination might help in finding ways to interfere with their formation, and ultimately prevent disease, by revealing the structure-function relationship and helping to design molecules targeting initial assembly steps and further propagation. Here, we describe the different steps in NMR protocols which allowed the 3D structure determination of amyloid-βfibrils.

Crystal structures, optical analysis and theoretical calculations of two thiosemicarbazide and semicarbazide derivatives based on triphenylamine-thiophene

Two new thiosemicarbazide and semicarbazide derivative based on triphenylamine-thiophene, (E)-2-((5-(4-(diphenylamino)phenyl)thiophen-2-yl)methylene)hydrazine-1-carboxamide (L1) and (E)-2-((5-(4-(diphenylamino)phenyl)thiophen-2-yl)methylene)hydrazine-1-carbothioamide (L2), were synthesized and fully characterized by IR, 1H NMR, 13C NMR, MS and single crystal X-ray structural determination. Their structures are similar, contained the same donor groups of triphenylaminyl units (TPA), the core thienyl ring (thienyl-C), and the =N-NH-CX-NH2 group (X = O, S) with different hetero atom. The absorption/emission maxima (in aspects of both wavelength and intensity) is altered by variation of the =N-NH-CX-NH2 groups. To clarify the influence mechanism of the hetero atom in photophysical properties, the computational results of them under state specific PCM/TD1 PBE1PBE method were performed in DCM solvent and illustrated by hole-electron transition method, which the excitation and de-excitation of them are illustrated by hole-electron transition method. Furthermore, ADCH charge indicated that the electron is transferred from the donor groups of TPA and thienyl-C to acceptor group (=N-NH-CX-NH2). The results illustrate that L2 transfers more electron from the donor groups to acceptor group (=N-NH-CS-NH2) than that (=N-NH-CO-NH2) of L1.

Screening and Research on Skin Barrier Damage Protective Efficacy of Different Mannosylerythritol Lipids.

Mannosylerythritol lipids (MELs) may prevent skin barrier damage, although their protective mechanisms and active monomeric constituents remain unclear. Here, three MELs were extracted from Candida antarctica cultures containing fermented olive oil then purified using silica gel-based column chromatography and semipreparative HPLC. All three compounds (MEL-A, MEL-B, MEL-C) were well separated and stable, and reliable materials were used for NMR and HRESIMS chemical structure determinations and for assessing MELs’ protective effects against skin damage. Notably, MEL-B and MEL-C effectively protected HaCaT cells from UVB-induced damage by upregulating the contents of filaggrin (FLG) and transglutaminase-1 (TGM1), as determined via ELISA. Moreover, MEL-B treatment (20 μg/mL) of UVB-irradiated HaCaT cells led to the upregulation of both the expression of mRNA genes and the key proteins FLG, LOR, and TGM1, which are known to be decreased in damaged skin cells. Additionally, histopathological analysis results revealed a markedly reduced intracellular vacuolation and cell damage, reflecting improved skin function after MEL-B treatment. Furthermore, immunofluorescence results revealed that MEL-B protected EpiKutis® three-dimensional cultured human skin cells from sodium dodecyl sulfate-induced damage by up-regulating FLG, LOR, and TGM1 expression. Accordingly, MELs’ protection against skin barrier damage depended on MEL-B monomeric constituent activities, thus highlighting their promise as beneficial ingredients for use in skin-care products.

SpecDB: A relational database for archiving biomolecular NMR spectral data

NMR is a valuable experimental tool in the structural biologist’s toolkit to elucidate the structures, functions, and motions of biomolecules. The progress of machine learning, particularly in structural biology, reveals the critical importance of large, diverse, and reliable datasets in developing new methods and understanding in structural biology and science more broadly. Biomolecular NMR research groups produce large amounts of data, and there is renewed interest in organizing these data to train new, sophisticated machine learning architectures and to improve biomolecular NMR analysis pipelines. The foundational data type in NMR is the free-induction decay (FID). There are opportunities to build sophisticated machine learning methods to tackle long-standing problems in NMR data processing, resonance assignment, dynamics analysis, and structure determination using NMR FIDs. Our goal in this study is to provide a lightweight, broadly available tool for archiving FID data as it is generated at the spectrometer, and grow a new resource of FID data and associated metadata. This study presents a relational schema for storing and organizing the metadata items that describe an NMR sample and FID data, which we call Spectral Database (SpecDB). SpecDB is implemented in SQLite and includes a Python software library providing a command-line application to create, organize, query, backup, share, and maintain the database. This set of software tools and database schema allow users to store, organize, share, and learn from NMR time domain data. SpecDB is freely available under an open source license at https://github.rpi.edu/RPIBioinformatics/SpecDB.

Resonance assignments toward solution-state NMR structure determination of novel box jellyfish protein

The box jellyfish eye lens protein, J2, belongs to the structural family known as crystallins, which form hydrogels to refract light and help contribute to varying levels of sight in different animals, ranging from rough light intensity to detailed images, although exactly how remains unknown. Current structure prediction software indicates that this crystallin has very little similarity to previously characterized proteins. This supports our hypothesis that it has a novel fold; potentially one of very few unique conformations identified in recent decades, which will be validated through structure determination.

Concise Modular Synthesis and NMR Structural Determination of Gallium Mycobactin T.

A modular synthesis of mycobactin T and its N-acetyl analogue is reported in a route that facilitates permutation of the lipid tails. A key feature is the generation of N(α)-Cbz-N(ε)-benzyloxy-N(ε)-Boc-lysine (A4) with methyl(trifluoromethyl)dioxirane in 59% yield. Selective hydroxamate N-acylation was achieved with acyl fluorides, enabling installation of lipids tails in the final step. O-Benzyl-dehydrocobactin T (B4) was prepared by modifying a known five-step sequence with an overall yield of 49%. 2-Hydroxyphenyl-4-carboxyloxazoline (C3) was prepared from 2-hydroxybenzoic acid and l-serine methyl ester in three steps with an overall yield of 55%. Ester coupling of A4 and B4 with EDCI afforded MbI-1 in 73% yield. Catalytic hydrogenation with Pd/BaSO4 and 50 psi of H2 simultaneously effected alkene reduction and debenzylation to afford MbI-2 in 96% yield. Fragment C3 was converted into acyl fluoride C4, which coupled with MbI-2 to afford MbI-3 in 51% yield. Finally, Boc-removal with HCl/EtOAc and treatment of the resultant hydroxylamine with stearyl fluoride furnished mycobactin T in 65% yield. Overall, the yield is 4% over 14 steps. The gallium mycobactin T-N-acetyl derivative (GaMbT-NAc) structure was determined by 1H NMR. The structure shows an octahedral Ga and two internal hydrogen bonds between peptidic N-Hs and two of the oxygen atoms coordinating Ga.

Unambiguous NMR Structural Determination of (+)-Catechin-Laccase Dimeric Reaction Products as Potential Markers of Grape and Wine Oxidation.

(+)-Catechin—laccase oxidation dimeric standards were hemi-synthesized using laccase from Trametes versicolor in a water-ethanol solution at pH 3.6. Eight fractions corresponding to eight potential oxidation dimeric products were detected. The fractions profiles were compared with profiles obtained with two other oxidoreductases: polyphenoloxidase extracted from grapes and laccase from Botrytis cinerea. The profiles were very similar, although some minor differences suggested possible dissimilarities in the reactivity of these enzymes. Five fractions were then isolated and analyzed by 1D and 2D NMR spectroscopy. The addition of traces of cadmium nitrate in the samples solubilized in acetone-d6 led to fully resolved NMR signals of phenolic protons, allowing the unambiguous structural determination of six reaction products, one of the fractions containing two enantiomers. These products can further be used as oxidation markers to investigate their presence and evolution in wine during winemaking and wine ageing.