15N Chemical Shifts Research Articles

Side chain 1H, 13C, 15N chemical shift assignments of lysine residues in delta+PHS/V66K staphylococcal nuclease selectively labeled with SAIL Lysine

Side chain 1H, 13C, 15N chemical shift assignments of lysine residues in delta+PHS/V66K staphylococcal nuclease selectively labeled with SAIL Lysine

NMR resonance assignments of the PR-10 allergens Act c 8 and Act d 8 from golden and green kiwifruit

Kiwifruits have become one of the most common food sources triggering allergic reactions. In patients suffering from birch pollen related food allergy, reactions result from initial sensitization to the birch (Betula verrucosa) pollen allergen Bet v 1, followed by immunological cross-reactivity to structurally homologous proteins in kiwifruit. Clinical symptoms range from scratching and itching of the oral cavity to more severe immunological reactions such as rhino conjunctivitis. In this work we assigned backbone and side chain 1H, 13C and 15N chemical shifts of the 17 kDa PR-10 allergens Act c 8.0101 and Act d 8.0101 from golden (Actinidia chinesis) and green (Actinidia deliciosa) kiwifruit by solution NMR spectroscopy. The chemical shift data confirm the characteristic Bet v 1 fold for both proteins, consisting of a seven-stranded antiparallel β-sheet interrupted by two short α-helices, along with a long C-terminal α-helix. Our data provide the basis for determining the three-dimensional solution structures of these proteins and characterizing their immunological cross-reactivity on a structural basis.

Backbone and methyl resonances assignment of the 87 kDa prefoldin from Pyrococcus horikoshii.

Prefoldin is a heterohexameric protein assembly which acts as a co-chaperonin for the well conserved Hsp60 chaperonin, present in archaebacteria and the eukaryotic cell cytosol. Prefoldin is a holdase, capturing client proteins and subsequently transferring them to the Hsp60 chamber for refolding. The chaperonin family is implicated in the early stages of protein folding and plays an important role in proteostasis in the cytosol. Here, we report the assignment of 1HN, 15N, 13C', 13Cα, 13Cβ, 1Hmethyl, and 13Cmethyl chemical shifts of the 87 kDa prefoldin from the hyperthermophilic archaeon Pyrococcus horikoshii, consisting of two α and four β subunits. 100% of the [13C, 1H]-resonances of Aβ, Iδ1, Iδ2, Tγ2, Vγ2 methyl groups were successfully assigned for both subunits. For the β subunit, showing partial peak doubling, 80% of the backbone resonances were assigned. In the α subunit, large stretches of backbone resonances were not detectable due to slow (μs-ms) time scale dynamics. This conformational exchange limited the backbone sequential assignment of the α subunit to 57% of residues, which corresponds to 84% of visible NMR signals.

Inclusion of the C-Terminal Domain in the β-Sheet Core of Heparin-Fibrillized Three-Repeat Tau Protein Revealed by Solid-State Nuclear Magnetic Resonance Spectroscopy.

Many neurodegenerative diseases such as Alzheimer's disease are characterized by pathological β-sheet filaments of the tau protein, which spread in a prion-like manner in patient brains. To date, high-resolution structures of tau filaments obtained from patient brains show that the β-sheet core only includes portions of the microtubule-binding repeat domains and excludes the C-terminal residues, indicating that the C-terminus is dynamically disordered. Here, we use solid-state NMR spectroscopy to identify the β-sheet core of full-length 0N3R tau fibrillized using heparin. Assignment of 13C and 15N chemical shifts of the rigid core of the protein revealed a single predominant β-sheet conformation, which spans not only the R3, R4, R' repeats but also the entire C-terminal domain (CT) of the protein. This massive β-sheet core qualitatively differs from all other tau fibril structures known to date. Using long-range correlation NMR experiments, we found that the R3 and R4 repeats form a β-arch, similar to that seen in some of the brain-derived tau fibrils, but the R1 and R3 domains additionally stack against the CT, reminiscent of previously reported transient interactions of the CT with the microtubule-binding repeats. This expanded β-sheet core structure suggests that the CT may have a protective effect against the formation of pathological tau fibrils by shielding the amyloidogenic R3 and R4 domains, preventing side-on nucleation. Truncation and post-translational modification of the CT in vivo may thus play an important role in the progression of tauopathies.

A Spectroscopic Overview of Intramolecular Hydrogen Bonds of NH…O,S,N Type.

Intramolecular NH…O,S,N interactions in non-tautomeric systems are reviewed in a broad range of compounds covering a variety of NH donors and hydrogen bond acceptors. 1H chemical shifts of NH donors are good tools to study intramolecular hydrogen bonding. However in some cases they have to be corrected for ring current effects. Deuterium isotope effects on 13C and 15N chemical shifts and primary isotope effects are usually used to judge the strength of hydrogen bonds. Primary isotope effects are investigated in a new range of magnitudes. Isotope ratios of NH stretching frequencies, νNH/ND, are revisited. Hydrogen bond energies are reviewed and two-bond deuterium isotope effects on 13C chemical shifts are investigated as a possible means of estimating hydrogen bond energies.

1H, 13C and 15N chemical shift assignments of the C-terminal domain of human UDP-Glucuronosyltransferase 2B7 (UGT2B7-C).

The human UDP-glucuronosyltransferase (UGT) family of enzymes catalyze the covalent addition of glucuronic acid to a wide range of compounds, generally rendering them inactive. Although important for clearance of environmental toxins and metabolites, UGT activation can lead to inappropriate glucuronidation of therapeutics underlying drug resistance. Indeed, 50% of medications are glucuronidated. To better understand this mode of resistance, we studied the UGT2B7 enzyme associated with glucuronidation of cancer drugs such as Tamoxifen and Sorafenib. We report 1H,13C and 15N backbone (> 90%) and side-chain assignments (~ 78% completeness according to CYANA) for the C-terminal domain of UGT2B7 (UGT2B7-C). Given the biomedical importance of this family of enzymes, our assignments will provide a key tool for improving understanding of the biochemical basis for substrate selectivity and other aspects of enzyme activity. This in turn will inform on drug design to overcome UGT-related drug resistance.

Partial magic angle spinning NMR 1H, 13C, 15N resonance assignments of the flexible regions of a monomeric alpha-synuclein: conformation of C-terminus in the lipid-bound and amyloid fibril states.

Alpha-synuclein (α-syn) is a small presynaptic protein that is believed to play an important role in the pathogenesis of Parkinson's disease (PD). It localizes to presynaptic terminals where it partitions between a cytosolic soluble and a lipid-bound state. Recent evidence suggests that α-syn can also associate with mitochondrial membranes where it interacts with a unique anionic phospholipid cardiolipin (CL). Here, we examine the conformation of the flexible fragments of a monomeric α-syn bound to lipid vesicles composed of anionic 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids, of tetraoleoyl CL (TOCL) and DOPC, and of fibrils. The dynamic properties of α-syn associated with DOPA:DOPC vesicles were the most favorable for conducting three-dimensional NMR experiments, and the 13C, 15N and amide 1H chemical shifts of the flexible and disordered C-terminus of α-syn could be assigned using three-dimensional through-bond magic angle spinning NMR spectroscopy. Although the C-terminus is more dynamically constrained in fibrils and in α-syn bound to TOCL:DOPC vesicles, a direct comparison of carbon chemical shifts detected using through bond two-dimensional spectroscopy indicates that the C-terminus is flexible and unstructured in all the three samples.

1H, 15N and 13C backbone and side-chain assignments of reduced and S-nitrosated C62only mutant of human thioredoxin.

Thioredoxins are ubiquitous and conserved small proteins. The redox-active site is composed of highly conserved Cys32 and Cys35. In higher eukaryotes, thioredoxin evolved to a gain of function in nitrosative control, with 3 extra cysteines, Cys62, Cys69, and Cys73. Human thioredoxin 1 (hTrx) is directly involved in cellular signal transduction through S-nitrosation. The understanding of the mechanism of S-nitrosation is essential. Here we produced a mutant of hTrx containing only Cys62 (C62only). We report the almost full 1H, 15N, and 13C chemical shift assignment of the reduced and S-nitrosated C62only. This study will help to measure the reactivity Cys62 toward S-nitrosants and the stability of S-nitrosated Cys62.

A “nucleophilic” iodine in a halogen-bonded iodonium complex manifests an unprecedented I+···Ag+ interaction

Summary When an electron is removed from a halogen atom, it forms a halenium ion X+ (X = I, Br, Cl). In halogen bonding (XB), X+ is considered as a strong XB donor, and when interacting with two XB acceptors (e.g., pyridine), it forms a halonium XB complex with a [N–I–N] three-center-four-electron bond with the two XB acceptors. An unprecedented I+···Ag+ interaction occurs between a [L1–I–L1]+ halogen-bonded complex and a [L2–Ag–L2]+ complex in which the iodonium ion acts like a nucleophile and donates electrons to the silver(I) cation. The X-ray diffraction analysis reveals a short contact [3.4608(3) A] between the I+ and Ag+ cations, and ITC measurements give a ΔG of −6.321 kcal/mol and Ka∼37,000 M−1 for the 1:1 complex. The DFT computational study on the nature of the I+···Ag+ interaction indicates that the I+ is nucleophilic in character, manifesting an unusual and strongly attractive interaction between the I+ and Ag+ cations.

1H,13C and 15N chemical shift assignments of the SUD domains of SARS-CoV-2 non-structural protein 3c: "The SUD-M and SUD-C domains".

SARS-CoV-2 RNA, nsP3c (non-structural Protein3c) spans the sequence of the so-called SARS Unique Domains (SUDs), first observed in SARS-CoV. Although the function of this viral protein is not fully elucidated, it is believed that it is crucial for the formation of the replication/transcription viral complex (RTC) and of the interaction of various viral “components” with the host cell; thus, it is essential for the entire viral life cycle. The first two SUDs, the so-called SUD-N (the N-terminal domain) and SUD-M (domain following SUD-N) domains, exhibit topological and conformational features that resemble the nsP3b macro (or “X”) domain. Indeed, they are all folded in a three-layer α/β/α sandwich structure, as revealed through crystallographic structural investigation of SARS-CoV SUDs, and they have been attributed to different substrate selectivity as they selectively bind to oligonucleotides. On the other hand, the C-terminal SUD (SUD-C) exhibit much lower sequence similarities compared to the SUD-N & SUD-M, as reported in previous crystallographic and NMR studies of SARS-CoV. In the absence of the 3D structures of SARS-CoV-2, we report herein the almost complete NMR backbone and side-chain resonance assignment (1H,13C,15N) of SARS-CoV-2 SUD-M and SUD-C proteins, and the NMR chemical shift-based prediction of their secondary structure elements. These NMR data will set the base for further understanding at the atomic-level conformational dynamics of these proteins and will allow the effective screening of a large number of small molecules as binders with potential biological impact on their function.

NMR assignments of vaccinia virus protein A28: an entry-fusion complex component.

Vaccinia virus (VACV) belonging to the poxvirus family enters the host cell via two different entry pathways; either endocytosis or virus/host cell membrane fusion. With respect to the virus/host cell membrane fusion, there are eleven viral membrane proteins forming a complicated entry-fusion complex (EFC), including A28, A21, A16, F9, G9, G3, H2, J5, L5, L1 and O3, to conduct the fusion function. These EFC components are highly conserved in all poxviruses and each of them is essential and necessary for the fusion activity. So far, with the exceptions of L1 and F9 whose crystal structures were reported, the structural information about other EFC components remains largely unclear. We aim to conduct a structural and functional investigation of VACV virus-entry membrane protein A28. In this work, we expressed and purified a truncated form of A28 (14kDa; residues 38-146, abbreviated as tA28 hereinafter), with deletion of its transmembrane domain (residues 1-22) and a hydrophobic segment (residues 23-37). And the assignments of its backbone and side chain 1H, 13C and 15N chemical shifts of tA28 are reported. The secondary structure propensity from TALOS+ indicates that tA28 does contain three α-helices, six β-strands and connecting loops. Aside from this, we demonstrated that tA28 does interact with fusion suppressor viral protein A26 (residues 351-500) by the 1H-15N HSQC spectrum. We interpret that A28 binding to A26 deactivates EFC fusion activity. The current study provides a valuable framework towards further structural analyses of this protein and for better understanding virus/host cell membrane fusion mechanism in association with virus entry.

1H, 13C, and 15N resonance assignments of reduced apo-WhiB4 from Mycobacterium tuberculosis.

The WhiB4 protein, a member of WhiB-like proteins, plays an important role in the survival and pathology of Mycobacterium tuberculosis (Mtb). As a transcription factor, WhiB4 regulates the expression of genes involved in maintaining redox homeostasis, central metabolism, and respiration. Furthermore, WhiB4 leads to the condensation of mycobacterial nucleoids and is capable of binding to DNA. WhiB4 contains four cysteine residues and exists in multiple forms under different redox environments, including a dimeric holo form with iron-sulfur cluster, multimeric disulfide-linked oxidized apo forms and monomeric reduced apo form. Here, we report the 1H, 13C, 15N chemical shifts of WhiB4 protein in its reduced apo state, providing a basis for the determination of its solution structure.

Modelling and simulation of 13C, 15N, 17O NMR chemical shifts, 17O and 14N electric field gradients and measurement of 13C and 15N chemical shifts in DNA/RNA of human gum cancer cells, tissues and tumors using NMR biospectroscopic profiling for novel systems diagnostics

Modelling and simulation of 13C, 15N, 17O NMR chemical shifts, 17O and 14N electric field gradients and measurement of 13C and 15N chemical shifts in DNA/RNA of human gum cancer cells, tissues and tumors using NMR biospectroscopic profiling for novel systems diagnostics

15N NMR studies provide insights into physico-chemical properties of room-temperature ionic liquids.

Ionic liquids (ILs) have gained a lot of attention as alternative solvents in many fields of science in the last two decades. It is known that the type of anion has a significant influence on the macroscopic properties of the IL. To gain insights into the molecular mechanisms responsible for these effects it is important to characterize these systems at the microscopic level. Such information can be obtained from nuclear spin-relaxation studies which for compounds with natural isotope abundance are typically performed using direct 1H or 13C measurements. Here we used direct 15N measurements to characterize spin relaxation of non-protonated nitrogens in imidazolium-based ILs which are liquid at ambient temperature. We report heteronuclear 1H-15N scalar coupling constants (nJHN) and 15N relaxation parameters for non-protonated nitrogens in ten 1-ethyl-3-methylimidazolium ([C2C1IM]+)-based ILs containing a broad range of anions. The 15N relaxation rates and steady-state heteronuclear 15N-{1H} NOEs were measured using direct 15N detection at 293.2 K and two magnetic field strengths, 9.4 T and 16.4 T. The experimental data were analyzed to determine hydrodynamic characteristics of ILs and to assess the contributions to 15N relaxation from 15N chemical shift anisotropy and from 1H-15N dipolar interactions with non-bonded protons. We found that the rotational correlation times of the [C2C1IM]+ cation determined from 15N relaxation measurements at room temperature correlate linearly with the macroscopic viscosity of the ILs. Depending on the selected anion, the 15N relaxation characteristics of [C2C1IM]+ differ considerably reflecting the influence of the anion on the physicochemical properties of the IL.

Structural basis for the binding of the cancer targeting scorpion toxin, ClTx, to the vascular endothelia growth factor receptor neuropilin-1.

Chlorotoxin (ClTx) is a 36-residue disulfide-rich peptide isolated from the venom of the scorpion Leiurus quinquestriatus. This peptide has been shown to selectively bind to brain tumours (gliomas), however, with conflicting reports regarding its direct cellular target. Recently, the vascular endothelial growth factor receptor, neuropilin-1 (NRP1) has emerged as a potential target of the peptide. Here, we sought to characterize the details of the binding of ClTx to the b1-domain of NRP1 (NRP1-b1) using solution state nuclear magnetic resonance (NMR) spectroscopy. The 3D structure of the isotope labelled peptide was solved using multidimensional heteronuclear NMR spectroscopy to produce a well-resolved structural ensemble. The structure points to three putative protein-protein interaction interfaces, two basic patches (R14/K15/K23 and R25/K27/R36) and a hydrophobic patch (F6/T7/T8/H10). The NRP1-b1 binding interface of ClTx was elucidated using 15N chemical shift mapping and included the R25/K27/R36 region of the peptide. The thermodynamics of binding was determined using isothermal titration calorimetry (ITC). In both NMR and ITC measurements, the binding was shown to be competitive with a known NRP1-b1 inhibitor. Finally, combining all of this data we generate a model of the ClTx:NRP1-b1 complex. The data shows that the peptide binds to the same region of NRP1 that is used by the SARS-CoV-2 virus for cell entry, however, via a non-canonical binding mode. Our results provide evidence for a non-standard NRP1 binding motif, while also providing a basis for further engineering of ClTx to generate peptides with improved NRP1 binding for future biomedical applications.

Backbone 1H, 15N, and 13C Chemical Shift Assignments for the Apo-State of Kemp Eliminase HG3.17

Backbone 1H, 15N, and 13C Chemical Shift Assignments for the Apo-State of Kemp Eliminase HG3.17

NMR resonance assignments of the FinO-domain of the RNA chaperone RocC

In prokaryotic species, gene expression is commonly regulated by small, non-coding RNAs (sRNAs). In the gram-negative bacterium Legionella pneumophila, the regulatory, trans-acting sRNA molecule RocR base pairs with a complementary sequence in the 5’-untranslated region of mRNAs encoding for proteins in the bacterial DNA uptake system, thereby controlling natural competence. Sense-antisense duplexing of RocR with targeted mRNAs is mediated by the recently described RNA chaperone RocC. RocC contains a 12 kDa FinO-domain, which acts as sRNA binding platform, along with an extended C-terminal segment that is predicted to be mostly disordered but appears to be required for repression of bacterial competence. In this work we assigned backbone and side chain 1H, 13C, and 15N chemical shifts of RocC’s FinO-domain by solution NMR spectroscopy. The chemical shift data for this protein indicate a mixed α/β fold that is reminiscent of FinO from Escherichia coli. Our NMR resonance assignments provide the basis for a comprehensive analysis of RocC’s chaperoning mechanism on a structural level.

13C and 15N chemical shift assignments of A117V and M129V human Y145Stop prion protein amyloid fibrils.

The C-terminally truncated Y145Stop variant of prion protein (PrP23-144) has been linked to a heritable prionopathy in humans and is also capable of triggering a transmissible prion disease in mice. PrP23-144 can be converted from soluble monomeric form to amyloid under physiological conditions, providing an in vitro model for investigating the molecular basis of amyloid strains and cross-seeding barriers. Here, we use magic-angle spinning solid-state NMR to establish the sequential backbone and sidechain 13C and 15N chemical shift assignments for amyloid fibrils formed by the A117V and M129V mutants of human PrP23-144, which in the context of full length PrP in vivo are among the specific residues associated with development of Gerstmann-Straüssler-Scheinker disease. The chemical shift data are utilized to identify amino acids comprising the rigid amyloid core regions and to predict the protein secondary structures for human PrP23-144 A117V and M129V fibrils.

Backbone 1H, 13C, and 15N Chemical Shift Assignments for C-terminal tail of human CRY1

Backbone 1H, 13C, and 15N Chemical Shift Assignments for C-terminal tail of human CRY1

A systematic benchmarking of 31P and 19F NMR chemical shift predictions using different DFT/GIAO methods and applying linear regression to improve the prediction accuracy

AbstractA systematic benchmark study of phosphorus and fluorine nuclear magnetic resonance chemical shift predictions using six different density functional theory (DFT)/the gauge‐including atomic orbital (GIAO) methods was conducted. Two databases were compiled: one consists of 35 phosphorus‐containing molecules, which cover the most common intramolecular bonding environments of trivalent and pentavalent phosphorus atoms; the other is composed of 46 fluorine‐containing molecules. The characteristics of each DFT/GIAO method with different solvent models were demonstrated in detail. The application of linear regression between the calculated isotropic shielding constants and experimental chemical shifts was applicable to improving the prediction accuracy. The best methods with the solvation model based on density (SMD) and conductor‐like polarizable continuum model (CPCM) implicit solvent models for 31P chemical shifts predictions are able to yield a root‐mean‐square deviation of 5.58 and 5.42 ppm, respectively; for 19F, the corresponding lowest prediction errors with these two applied solvent models are 4.43 and 4.12 ppm, respectively. The developed scaling factors fitted from linear regression are applicable to enhancing the chance of successful structural elucidations of phosphorus or fluorine‐containing compounds as an efficient complement to 13C, 1H, 11B, and 15N chemical shift predictions.