Resonance Assignments Research Articles

1H, 15N and 13C backbone resonance assignment of the N-terminal region of Zika virus NS4B protein in detergent micelles.

Zika virus has raised global concerns due to its link to microcephaly and Guillain-Barré syndrome in adults. One of viral nonstructural proteins-NS4B, an integral membrane protein, plays crucial roles in viral replication by interacting with both viral and host proteins, rendering it an attractive drug target for antiviral development. We purified the N-terminal region of ZIKV NS4B (NS4B NTD) and reconstituted it into detergent micelles. Here, we report the assignments of the backbone resonances of NS4B NTD in detergent micelles. The available assignment is useful for understanding its structure and ligand binding to provide useful information for developing NS4B inhibitors.

Backbone 1H, 15N, and 13C resonance assignments of the FF1 domain from P190A RhoGAP in 5 and 8M urea.

The Rho GTPase (Ras homolog GTPases) system is a crucial signal transducer that regulates various cellular processes, including cell cycle and migration, genetic transcription, and apoptosis. In this study, we investigated the unfolded state of the first FF domain (FF1) of P190A RhoGAP, which features four tandem FF domains. For signal transduction, FF1 is phosphorylated at tyrosine 308 (Y308), which is buried in the hydrophobic core and is inaccessible to kinases in the folded domain. It was proposed, therefore, that the phosphorylation occurs in a transiently populated unfolded state of FF1. To probe the folding pathway of the RhoGAP FF1 domain, here we have performed a nearly complete backbone resonance assignments of a putative partially unfolded state of FF1 in 5M urea and its fully unfolded state in 8M urea.

Optimization of 15N–13C double-resonance NMR experiments under low temperature magic angle spinning dynamic nuclear polarization conditions

Dynamic nuclear polarization (DNP) enhanced magic angle spinning (MAS) solid-state NMR carried out at 25 K enables rapid acquisition of multi-dimensional 13C–15N correlation spectra for protein structure studies and resonance assignment. Under commonly used DNP conditions, solvent deuteration reduces 1H–15N cross polarization (CP) efficiencies, necessitates more careful optimization, and requires longer high-power 15N radio-frequency pulses. The sensitivity of 2D heteronuclear correlation experiments is potentially impaired. Here we show that 2D 15N-13C experiments based on 13C-15N transferred echo double resonance (TEDOR) methods outperform 2D experiments based on CP transfers in a fully deuterated solvent, and are competitive with CP-based experiments when the solvent is only partially deuterated. Additionally, we show that optimization of TEDOR-based 2D experiments is simpler than optimization of CP-based experiments under 25 K MAS conditions.

Effect of Chlorination on the Shape Resonance Spectrum of CH2Br2 and CCl2Br2 Molecules in Collisions with Electrons.

In this work, we present a theoretical study of elastic electron scattering by the CH2Br2 and CCl2Br2 molecules using the Schwinger multichannel method. Through the scattering amplitudes computed at the static-exchange and static-exchange plus polarization levels of approximation, we have obtained integral, momentum transfer, and differential cross sections for energies ranging from 0 to 20 eV. For both systems, the resonance spectrum was identified in the cross sections and characterized with basis on the analysis of the eigenvalues and eigenvectors obtained by diagonalization of the scattering Hamiltonian. Despite some discrepancies, our cross sections and resonance assignments are in overall good agreement with the experimental and theoretical data available in the literature. Present results also indicate that the effect of chlorination (i.e., the substitution of the hydrogen atoms by chlorine atoms in going from CH2Br2 to CCl2Br2) on the scattering process causes the following outcomes: the change in the position of the resonances in the low-energy regime and the increase in the magnitude of the cross sections for higher energies.

1H, 15N and 13C resonance assignments of the S2A and H64A double mutant of human carbonic anhydrase II.

Protein-water interactions profoundly influence protein structure and dynamics. Consequently, the function of many biomacromolecules is directly related to the presence and exchange of water molecules. While structural water molecules can be readily identified through X-ray crystallography, the dynamics within functional protein-water networks remain largely elusive. Therefore, to understand the role of biological water in protein dynamics and function, we have introduced S2A and H64A mutations in human Carbonic Anhydrase II (hCAII), a model system to study protein-water interactions. The mutations of serine to alanine at position 2 and histidine to alanine at position 64 cause an increase in hydrophobicity in the N-terminus and active site loop thereby restricting water entry and disrupting the water network in the Zn2+-binding pocket. To pave the way for a detailed investigation into the structural, functional, and mechanistic aspects of the Ser2Ala/His64Ala double mutant of hCAII, we present here almost complete sequence-specific resonance assignments for 1H, 15N, and 13C. These assignments serve as the basis for comprehensive studies on the dynamics of the protein-water network within the Zn2+-binding pocket and its role in catalysis.

Chemical shift assignments of PA2072 CHASE4 domain.

Diverse extracellular sensor domains enable cells to regulate their behavior, physiological processes, and interspecies interactions in response to environmental stimuli. These sensing mechanisms facilitate the ultimate adaptation of organisms to their surrounding conditions. Pseudomonas aeruginosa (PAO1) is a clinically significant opportunistic pathogen in hospital infection. The CHASE4 domain, a putative extracellular sensing module, is found in the N-terminus of GGDEF-EAL-containing PA2072, a transmembrane receptor from P. aeruginosa. However, the signal identification and sensing mechanism of monomeric PA2072 CHASE4 remains largely unknown. Here, we report backbone and side chain resonance assignments of PA2072 CHASE4 as a basis for studying the structural mechanism of CHASE4-mediated signal recognition.

Backbone assignment of CcdB_G100T toxin from E.coli in complex with the toxin binding C-terminal domain of its cognate antitoxin CcdA.

The CcdAB system expressed in the E.coli cells is a prototypical example of the bacterial toxin-antitoxin (TA) systems that ensure the survival of the bacterial population under adverse environmental conditions. The solution and crystal structures of CcdA, CcdB and of CcdB in complex with the toxin-binding C-terminal domain of CcdA have been reported. Our interest lies in the dynamics of CcdB-CcdA complex formation. Solution NMR studies have shown that CcdB_G100T, in presence of saturating concentrations of CcdA-c, a truncated C-terminal fragment of CcdA exists in equilibrium between two major populations. Sequence specific backbone resonance assignments of both equilibrium forms of the ~ 27kDa complex, have been obtained from a suite of triple resonance NMR experiments acquired on 2H, 13C, 15N enriched samples of CcdB_G100T. Analysis of 1H, 13Cα, 13Cβ secondary chemical shifts, shows that both equilibrium forms of CcdB_G100T have five beta-strands and one alpha-helix as the major secondary structural elements in the tertiary structure. The results of these studies are presented below.

The backbone NMR resonance assignments of the stabilized E. coli β clamp.

The 81 kDa E. coli β clamp is a ring-shaped head-to-tail homodimer that encircles DNA and plays a central role in bacterial DNA replication by serving as a processivity factor for DNA polymerases and a binding platform for other DNA replication and repair proteins. Here we report the backbone 1H, 15N, and 13C NMR resonance assignments of the stabilized T45R/S107R β clamp variant obtained using standard TROSY-based triple-resonance experiments (BMRB 52548). The backbone assignments were aided by 13C and 15N edited NOESY experiments, allowing us to utilize our previously reported assignments of the β clamp ILV side-chain methyl groups (BMRB 51430, 51431). The backbone assignments of the T45R/S107R β clamp variant were transferred to the wild-type β clamp using a minimal set of TROSY-based 15N edited NOESY, NHCO and NHCA experiments (BMRB 52549). The reported backbone and previous ILV side-chain resonance assignments will enable NMR studies of the β clamp interactions and dynamics using amide and methyl groups as probes.

The 1H, 15N, and 13C resonance assignments of a single-domain antibody against immunoglobulin G.

Research on camelid-derived single-domain antibodies (sdAbs) has demonstrated their significant utility in diverse biotechnological applications, including therapy and diagnostic. This is largely due to their relative simplicity as monomeric proteins, ranging from 12 to 15kDa, in contrast to immunoglobulin G (IgG) antibodies, which are glycosylated heterotetramers of 150-160kDa. Single-domain antibodies exhibit high conformational stability and adopt the typical immunoglobulin domain fold, consisting of a two-layer sandwich of 7-9 antiparallel beta-strands. They contain three loops, known as complementary-determining regions (CDRs), which are assembled on the sdAb surface and are responsible for antigen recognition. The single-domain antibody examined in this study, sdAb-mrh-IgG, was engineered to recognize IgG from rats, mice, but it also weakly recognizes IgG from humans (Pleiner et al. 2018). A search of the Protein Data Bank revealed only one NMR structure of a single-domain antibody, which is unrelated to sdAb-mrh-IgG. The NMR chemical shift assignments of sdAb-mrh-IgG will be utilized to study its molecular dynamics and interactions with antigens in solution, which is fundamental for the rational design of novel single-domain antibodies.

NMR 1H, 13C, 15N backbone resonance assignments of 14-3-3ζ binding region of human FOXO3a (residues 1-284)

In tumors, mutation in Ras proteins stimulates a signaling cascade through phosphorylation. Downstream of the cascade, many transcription and translation factors are up- or down-regulated by phosphorylation, leading to cancer progression. This phosphorylation cascade is sustained by 14-3-3ζ protein. 14-3-3ζ binds to its client proteins that are Ser/Thr-phosphorylated and prevents their dephosphorylation. One of those transcription factors is FOXO3a, whose transcriptional activity is suppressed in the phosphorylation cascade. FOXO3a binds to specific DNA sequences and activates the transcription of apoptosis-related proteins. In cancer cells, however, FOXO3a is phosphorylated, bound to 14-3-3ζ, and dissociated from the DNA, resulting in FOXO3a inactivation. To elucidate the mechanism of FOXO3a inactivation by the 14-3-3ζ binding, we aim to perform NMR analysis of the interaction between 14-3-3ζ and di-phosphorylated FOXO3a residues 1-284 (dpFOXO3a). Here, we report the backbone resonance assignments of dpFOXO3a, which are transferred from those of the N-terminal domain (NTD) and the DNA-binding domain (DBD) of dpFOXO3a.

1H, 13C and 15N backbone resonance assignment of the calcium-activated EndoU endoribonuclease.

The catalytic domain of the calcium-dependent endoribonuclease EndoU from Homo sapiens was expressed in E. coli with 13C and 15N labeling. A nearly complete assignment of backbone 1H, 15N, and 13C resonances was obtained, as well as a secondary structure prediction based on the assigned chemical shifts. The predicted secondary structures were almost identical to the published crystal structure of calcium-activated EndoU. This is the first NMR study of an eukaryotic member of the EndoU-like superfamily of ribonucleases.

NMR-based solution structure of the Caulobacter crescentus ProXp-ala trans-editing enzyme

ProXp-ala is a key component of the translational machinery in all three Domains of life. This enzyme helps to maintain the fidelity of proline codon translation through aminoacyl-tRNAPro proofreading. In the first step of tRNA aminoacylation, the cognate aminoacyl-tRNA synthetase (aaRS) binds and activates an amino acid in the enzyme’s synthetic active site. If a non-cognate amino acid passes this first selection step and is charged onto the tRNA, a distinct aaRS editing active site may recognize the mischarged tRNA and deacylate it. Alternatively, this editing reaction may be carried out by a separate enzyme that deacylates the mischarged tRNA in trans. ProXp-ala is responsible for editing Ala mischarged onto tRNAPro. Since trans-editing domains such as ProXp-ala bind their substrates after release from the synthetase, they must recognize not only the mischarged amino acid, but also the specific tRNA. Previous studies showed that Caulobacter crescentus (Cc) ProXp-ala distinguishes tRNAPro from tRNAAla, in part, based on the unique tRNAPro acceptor stem base pair C1:G72. Previous crystallographic and NMR data also revealed a role for conformational selection by the ProXp-ala α2 helix in Ala- versus Pro-tRNAPro substrate discrimination. The α2 helix makes lattice contacts in the crystal, which left some uncertainty as to its position in solution. We report resonance assignments for the substrate-free Cc ProXp-ala and the NMR-derived three-dimensional structure of the protein. These data reveal the position of the α2 helix in solution, with implications for substrate binding and recognition.

Solution NMR backbone resonance assignment of the full-length resistance-related calcium-binding protein Sorcin.

Sorcin is a penta-EF hand calcium-binding protein that confers multidrug resistance in cancer cells. It regulates cellular Ca2+ homeostasis by interacting with calcium channels such as Ryanodine receptor 2 and Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase in a calcium-dependent manner. The crystal structure of the Sorcin has been determined in both calcium-free and calcium-bound states to understand calcium-binding induced conformational change. However, due to its flexibility, most of the N-terminal domain is invisible in these crystal structures. Here we report the 1H, 13C, and 15N backbone resonance assignments of full-length Sorcin in the calcium-free state using solution NMR. The protein secondary structure was predicted based on the assigned backbone chemical shifts using TALOS+ and CSI 3.0. Our backbone resonance assignment of the full-length Sorcin provides a foundation for future NMR spectroscopic studies to uncover the mechanism of Ca2+ sensing by Sorcin.

1H, 15N and 13C resonance assignments of eggcase silk protein 3.

Spider silk is a high-performance biomaterial known for its outstanding combination of strength and flexibility. Among the six distinct types of spider silk, eggcase silk stands out as it is exclusively produced from the tubuliform gland, playing a specialized role in offspring protection. In the spider species Latrodectus hesperus, eggcase silk is spun from a large spidroin complex, including the major silk component tubuliform spidroin 1 (TuSp1) and at least six different minor silk components. One of these minor components is eggcase protein 3 (ECP3), a small silk protein of 11.8kDa that lacks the typical spidroin architecture. ECP3 shows very limited homology to all known spidroins. In this study, we report nearly complete backbone and side-chain resonance assignments of ECP3 as a basis for studying the structural mechanisms involved in eggcase silk formation.

Experimental and Computational 77Se NMR Spectroscopic Study on Selenaborane Cluster Compounds.

Calculated and measured 77Se nuclear magnetic resonance (NMR) chemical shift data on a diverse collection of 13 selenaborane cluster compounds, containing a total of 19 selenium centers, reveals a correlation between chemical shifts and the intracluster coordination of selenium atoms within their borane frameworks. A plot of the measured against calculated 77Se NMR chemical shifts shows an approximately linear relationship that can serve as a predictive tool in assessing the chemical shift range in which a selenium vertex from a particular compound might be expected to be found, thereby reducing expensive experimental time. Furthermore, the relative chemical shifts between selenium vertices in clusters containing more than one selenium atom are consistent across the range, thus allowing the assignment of the selenium resonances with a high degree of confidence even in relatively low-level density functional theory calculations. A new macropolyhedral 20-vertex selenaborane Se2B18H20 (A) is also reported.

1H, 13C and 15N resonance assignments of a shark variable new antigen receptor against hyaluronan synthase.

Single domain antibody (sdAb) is only composed of a variable domain of the heavy-chain-only antibody, which is devoid of light chain and naturally occurring in camelids and cartilaginous fishes. Variable New Antigen Receptor (VNAR), a type of single domain antibody present in cartilaginous fishes such as sharks, is the smallest functional antigen-binding fragment found in nature. The unique features, including flexible paratope, high solubility and outstanding stability make VNAR a promising prospect in antibody drug development and structural biology research. However, VNAR's research has lagged behind camelid-derived sdAb, especially in the field of structural research. Here we report the 1H,15N,13C resonance assignments of a VNAR derived from the immune library of Chiloscyllium plagiosum, termed B2-3, which recognizes the hyaluronan synthase. Analysis of the backbone chemical shifts demonstrates that the secondary structure of VNAR is predominately composed of β-sheets corresponding to around 40% of the B2-3 backbone. The Cβ chemical shift values of cysteine residues, combined with mass spectrometry data, clearly shows that B2-3 contains two pairs of disulfide bonds, which is import for protein stability. The assignments will be essential for determining the high resolution solution structure of B2-3 by NMR spectroscopy.

From mother's milk to structural insights: 1H–15N NMR analysis of Lewis X antigen-bearing oligosaccharides isolated from human milk

Human milk oligosaccharides (HMOs) are complex unconjugated glycans abundant in breast milk, with vital roles in infant nutrition and health. The structural elucidation of HMOs remains challenging due to their diverse compositions and isomeric complexities. This study presents a novel approach employing 1H–15N NMR correlations to pinpoint and characterize HMOs, LNH, LNnH, and their fucosylated derivatives including those of bearing the Lewis X motif. Isolated from human milk, these hexa-, hepta-, and octasaccharides were extensively analysed by NMR. This enabled their first complete 1H, 13C and 15N resonance assignments. Using 1H–15N HSQC experiments, the distinctive 1H–15N correlations of GlcNAc units within the HMOs provided structural fingerprints, allowing for unequivocal differentiation of isomeric structures and fucosylation patterns. Results demonstrate the potential of 1H–15N NMR spectroscopy in decoding complex HMO structures, offering new perspectives on their detailed structural characterization and contributing to a deeper understanding of their potential benefits.

Bioproduction Platform to Generate Functionalized Disulfide-Constrained Peptide Analogues.

Disulfide-constrained peptides (DCPs) have gained increased attention as a drug modality due to their exceptional stability and combined advantages of large biologics and small molecules. Chemical synthesis, although widely used to produce DCPs, is associated with high cost, both economically and environmentally. To reduce the dependence on solid phase peptide synthesis and the negative environmental footprint associated with it, we present a highly versatile, low-cost, and environmentally friendly bioproduction platform to generate DCPs and their conjugates as well as chemically modified or isotope-labeled DCPs. Using the DCP against the E3 ubiquitin ligase Zinc and Ring Finger 3, MK1-3.6.10, as a model peptide, we have demonstrated the use of bacterial expression, combined with Ser ligation or transglutaminase-mediated XTEN ligation, to produce multivalent MK1-3.6.10 and MK1-3.6.10 with N-terminal functional groups. We have also developed a bioproduction method for the site-specific incorporation of unnatural amino acids into recombinant DCPs by the amber codon suppression system. Lastly, we produced 15N/13C-labeled MK1-3.6.10 with high yield and assessed the performance of a semiautomated resonance assignment workflow that could be used to accelerate binding studies and structural characterization of DCPs. This study provides a proof of concept to generate functionalized DCPs using bioproduction, providing a potential solution to alleviate the reliance on hazardous chemicals, reduce the cost, and expedite the timeline for DCP discovery.

NMR side-chain assignments of the Crimean–Congo hemorrhagic fever virus glycoprotein n cytosolic domain

Abstract. We assigned the side-chain resonances of the Crimean–Congo hemorrhagic fever virus (CCHFV) glycoprotein n (Gn) cytosolic domain that is 69 amino acids long to complete the backbone resonances previously published by Estrada et al. (2011). The process was facilitated by three factors. First, sample preparation using cell-free protein synthesis (CFPS) was completed in less than 2 d and allowed for correct zinc finger formation by adding zinc ions to the reaction. Second, access to NMR platforms with standardized pulse sequences allowed for data acquisition in 18 d. Third, data analysis using the online platform NMRtist allowed sequential resonance assignments to be made in a day, and assignments were verified and finalized in less than a week. Our work thus provides an example of how NMR assignments, including side chains, of small and well-behaved proteins can be approached in a rapid routine, at protein concentrations of 150 µM.

Backbone and methyl side-chain resonance assignments of the Fab fragment of adalimumab

Adalimumab is a therapeutic monoclonal antibody developed to target human TNF an important mediator of immune-mediated inflammatory diseases such as rheumatoid arthritis, amongst others. The 48 kDa Fab fragment of adalimumab was produced in Escherichia coli using a single chain approach to allow complete isotopic incorporation of deuterium, carbon-13 and nitrogen-15 along with the protonated isoleucine-d, valine and leucine methyl groups. Here we report the near complete resonance assignment of the polypeptide backbone and the methyl groups of isoleucine, leucine and valine residues.