Solution Conformation Research Articles

The antimicrobial peptide chensinin-1b alleviates the inflammatory response by targeting the TLR4/NF-κB signaling pathway and inhibits Pseudomonas aeruginosa infection and LPS-mediated sepsis

Excessive inflammatory responses are a major contributor to the high mortality associated with sepsis, a prevalent global complication. Therefore, the potential therapeutic strategy for sepsis involves targeting macrophages and reducing proinflammatory cytokine release. Chensinin-1b, an analog of the natural antimicrobial peptide derived from Rana chensinensis skin secretion, exhibits broad-spectrum antibacterial activity and adopts a random coil conformation in both PBS and membrane solution. By efficiently neutralizing LPS, chensinin-1b holds promise in alleviating LPS-induced inflammatory responses. In this study, we established a mouse septic shock model by exposing mice to multiple-drug-resistant Pseudomonas aeruginosa, as well as an endotoxin-mediated sepsis model induced by LPS. Administering chensinin-1b significantly prolonged the survival of the experimental mice, concurrently mitigating inflammatory responses and reducing organ damage. Additionally, we investigated the anti-inflammatory mechanism of chensinin-1b using a constructed LPS-induced mouse macrophage RAW264.7 inflammatory model. Our findings demonstrated that chensinin-1b effectively mitigated the excessive activation of the TLR4/NF-κB signaling pathway by directly neutralizing extracellular LPS, thus ameliorating the inflammatory response. Moreover, upon blocking the TLR4 signaling pathway, chensinin-1b further reduced the release of proinflammatory cytokines induced by LPS, indicating alternative modes of regulation. Notably, chensinin-1b rapidly entered RAW264.7 cells within 30 min via endocytosis, diffusing into the cytoplasm while retaining its anti-inflammatory properties intracellularly. Although further investigations are warranted to comprehensively elucidate the intracellular anti-inflammatory mechanism of chensinin-1b, our findings substantiate its possession of anti-inflammatory properties both intracellularly and extracellularly. Thus, chensinin-1b emerges as a promising candidate for mitigating excessive inflammatory responses associated with sepsis.

Dectin-1 multimerization and signaling depends on fungal β-glucan structure and exposure

Dectin-1A is a C-type lectin innate immunoreceptor that recognizes β-(1,3;1,6)-glucan, a structural component of Candida species cell walls. β-Glucans can adopt solution structures ranging from random coil to insoluble fiber due to tertiary (helical) and quaternary structure. Fungal β-glucans of medium and high molecular weight are highly structured, but low molecular weight glucan is much less structured. Despite similar affinity for Dectin-1, the ability of glucans to induce Dectin-1A-mediated signaling correlates with degree of structure. Glucan denaturation experiments showed that glucan structure determines agonistic potential, but not receptor binding affinity. We explored the impact of glucan structure on molecular aggregation of Dectin-1A. Stimulation with glucan signaling decreased Dectin-1A diffusion coefficient. Fluorescence measurements provided direct evidence of ligation-induced Dectin-1A aggregation, which positively correlated with increasing glucan structure content. In contrast, Dectin-1A is predominantly in a low aggregation state in resting cells. Molecular aggregates formed during interaction with highly structured, agonistic glucans did not exceed relatively small (<15 nm) clusters of a few engaged receptors. Finally, we observed increased molecular aggregation of Dectin-1A at fungal particle contact sites in a manner that positively correlated with the degree of exposed glucan on the particle surface. These results indicate that Dectin-1A senses the solution conformation of β-glucans through their varying ability to drive receptor dimer/oligomer formation and activation of membrane proximal signaling events.

Evaluation of nonnatural L-iminosugar C,C-glycosides, a new class of C-branched iminosugars, as glycosidase inhibitors

Capitalizing on a previously developed Staudinger/azaWittig/Grignard (SAWG)-ring contraction sequence that furnished protected six-membered L-iminosugar C,C-glycosides bearing an allyl group and various substituents at the pseudoanomeric position, the synthesis and glycosidase inhibition of a small library of six- and seven-membered L-iminosugar C,C-glycosides is reported. Their hydrogenolysis or cyclization by RCM followed by deprotection afforded eleven L-iminosugars including spirocyclic derivatives. All compounds adopt a 1C4 conformation in solution according to NMR data. Compared to previously reported branched L-iminosugars, the L-iminosugar C,C-glycosides reported herein were less potent glycosidase inhibitors. However, some of these compounds showed micromolar inhibition of human lysosome β-glucocerebrosidase suggesting that such iminosugars could be useful to access potent CGase inhibitors by adjusting the structure/length of the pseudoanomeric substituents.

Conformationally constrained cyclic grafted peptidomimetics targeting protein-protein interactions.

Sunflower trypsin inhibitor-1 (SFTI-1) structure is used for designing grafted peptides as a possible therapeutic agent. The grafted peptide exhibits multiple conformations in solution due to the presence of proline in the structure of the peptide. To lock the grafted peptide into a major conformation in solution, a dibenzofuran moiety (DBF) was incorporated in the peptide backbone structure, replacing the Pro-Pro sequence. NMR studies indicated a major conformation of the grafted peptide in solution. Detailed structural studies suggested that SFTI-DBF adopts a twisted beta-strand structure in solution. The surface plasmon resonance analysis showed that SFTI-DBF binds to CD58 protein. A model for the protein-SFTI-DBF complex was proposed based on docking studies. These studies suggested that SFTI-1 grafted peptide can be used to design stable peptides for therapeutic purposes by grafting organic functional groups and amino acids. However, when a similar strategy was used with another grafted peptide, the resulting peptide did not produce a single major conformation, and its biological activity was lost. Thus, conformational constraints depend on the sequence of amino acids used for SFTI-1 grafting.

Unmasking the True Identity of Rapamycin's Minor Conformer.

Rapamycin, a well-known macrocyclic natural product with myriad biological activities, has been the subject of intense study since its first isolation and characterization over five decades ago. Rapamycin has been found to adopt a single conformation in the solid state (both when protein bound and uncomplexed) and exists as a mixture of two conformations in solution. Early work established that the major conformer in solution is the trans amide isomer but left the minor conformer mostly uncharacterized. Since that time, it has been widely accepted that the minor conformer of rapamycin is the cis amide, based solely on analogy to FK-506, another potent immunosuppressive compound with some shared key structural elements. To address this long-standing and unresolved question, the solution structure of the minor conformer of rapamycin was investigated using a combination of NMR techniques and computational methods and determined to be a trans amide species with rotation about the ester linkage.

Resolving Hidden Solution Conformations of Hemoglobin Using IMS-IMS on a Cyclic Instrument.

Ion mobility spectrometry-mass spectrometry (IMS-MS) experiments on a cyclic IMS instrument were used to examine heterogeneous distributions of structures found in the 15+ to 18+ charge states of the hemoglobin tetramer (Hb). The resolving power of IMS measurements is known to increase with increasing drift-region length. This effect is not significant for Hb charge states as peaks were shown to broaden with increasing drift-region length. This observation suggests that multiple structures with similar cross sections may be present. To examine this hypothesis, selections of drift time distributions were isolated and subsequently reinjected into the mobility region for additional separation. These IMS-IMS experiments demonstrate that selected regions separate further upon additional passes around the drift cell, consistent with the idea that initial resolving power was limited due to the presence of many closely related conformations. Additional variable temperature electrospray ionization (vT-ESI) experiments were conducted to study how changing the solution temperature affects solution conformations. Some features in these IMS-IMS studies were observed to change similarly with solution temperature compared to features in the single IMS distribution. Other features changed differently in the selected mobility data, indicating that solution structures that were obscured upon IMS analysis because of the complex heterogeneity of the original distribution are discernible after reducing the number of conformers that are analyzed by further IMS analysis. These results illustrate that the combination of vT-ESI with IMS-IMS is useful for resolving and exploring conformer distributions and stabilities in systems that exhibit a large degree of structural heterogeneity.

Helices in Peptides Containing Differentially Geminally Di‐substituted γ Amino Acid Residues

AbstractWe have explored the relative propensity of the three differentially di‐substituted γ amino acid residues, to be accommodated in all α helical conformations. We have established that small chiral hybrid peptides Boc‐Aib‐γx,x‐Aib‐Leu‐Aib‐Val‐OMe (x,x=2,2/3,3/4,4) containing single geminally di‐substituted γ amino acid residue, can adopt both handedness of helical conformation in crystals and solution. The relative ease of being accommodated in helical structures decreased in the order γ4,4&gt;γ3,3&gt;γ2,2. γ4,4 and γ3,3 readily adopted C12 helical conformations unlike γ2,2. γ2,2 and γ3,3 containing peptides, adopted either handedness of the helical conformation in crystals while in solution, they adopted right‐handed helical conformation. Peptide, containing γ4,4 only adopted right‐handed helical conformation both in the solid and solution states. Our studies established that position of di‐substitution along the backbone of γ amino acid residues determined their conformational preference, and thus might be used to generate diversity in the peptide conformations.

Desolvation Induced Conformational Transition in a Cryptophane Host: Crystal To Crystal Transformation.

Cryptophanes show different conformations in solution and solid state depending upon various factors, such as the length of connecting linkers, medium, and nature of the incoming guest molecule(s). A cyclotriguaiacylenes (CTG) based cryptophane molecule was synthesized using click chemistry containing three triazole linkers and studied as well. This molecule shows two conformations, out-out crown-crown (CC), and out-in CC, in the presence or absence of guest molecule(s), as studied both in solution and solid state. The out-in CC, in which both CTG fragments are in crown conformation with one crown sitting above the other, could be obtained by slow escape of the trapped acetone molecules from out-out CC in solid state. This transformation could be obtained through a single-crystal-to-single-crystal (SCSC) transformation from a large volume out-out CC to a smaller volume out-in CC conformation which is also supported by density functional theory calculations.

Surface morphology and action mechanism of macrophage immunostimulating activity of Rosa Setate x Rosa Rugosa polysaccharide WSRP-1b

Our previous research demonstrated that the glucomannan WSRP-1b, which was obtained from waste biomass of Rosa Setate x Rosa Rugosa, had potent immunostimulatory activity. However, the specific mechanisms involved in the activation of RAW264.7 macrophages by WSRP-1b remain unclear. Scanning electron microscopy, atomic force microscopy and Congo red analyses revealed that WSRP-1b exhibited a flake structure with irregular curls and had a triple-helix conformation in solution. The immunostimulation assays showed that WSRP-1b could substantially enhance macrophages phagocytosis and promote the accumulation of iNOS, TNF-α, and IL-6 mRNA. Western blot analysis indicated that the expression of mitogen-activated protein kinases (MAPKs) was upregulated in WSRP-1b-induced RAW264.7 cells. Furthermore, pretreatment of RAW 264.7 cells using specific inhibitors against MAPKs and NF-κB remarkably attenuated nitric oxide production and cytokine secretion. These data suggest that WSRP-1b activates RAW264.7 macrophages via the MAPK and NF-κB signaling pathways and may be a novel immunomodulatory agent for hypoimmunity disease therapy.

In-droplet hydrogen-deuterium exchange to examine protein/peptide solution conformer heterogeneity.

Many different structure analysis techniques are not capable of probing the heterogeneity of solution conformations. Here, we examine the ability of in-droplet hydrogen-deuterium exchange (HDX) to directly probe solution conformer heterogeneity of a protein with mass spectrometry (MS) detection. Two vibrating capillary vibrating sharp-edge spray ionization (cVSSI) devices have been arranged such that they generate microdroplet plumes of the analyte and D2 O reagent, which coalesce to form reaction droplets where HDX takes place in the solution environment. The native HDX-MS setup has been first explored for two model peptides that have distinct structural compositions in solution. The effectiveness of the multidevice cVSSI-HDX in illustrating structural details has been further exploited to investigate coexisting solution-phase conformations of the protein ubiquitin. In-droplet HDX reveals decreased backbone exchange for a model peptide that has a greater helix-forming propensity. Differences in intrinsic rates of the alanine and serine residues may account for much of the observed protection. The data allow the first estimates of backbone exchange rates for peptides undergoing in-droplet HDX. That said, the approach may hold greater potential for investigating the tertiary structure and structural transitions of proteins. For ubiquitin protein, HDX reactivity differences suggest that multiple conformers are present in native solutions. The addition of methanol to buffered aqueous solutions of ubiquitin results in increased populations of solution conformers of higher reactivity. Data analysis suggests that partially folded conformers such as the A-state of ubiquitin increase with methanol content; the native state may be preserved to a limited degree even under stronger denaturation conditions. The deuterium uptake after in-droplet HDX has been observed to correspond to some degree with peptide backbone hydrogen protection based on differences in intrinsic rates of exchange. The presence of coexisting protein solution structures under native and denaturing solution conditions has been distinguished by the isotopic distributions of deuterated ubiquitin ions.

Ladder and zig-zag Feynman diagrams, operator formalism and conformal triangles

We develop an operator approach to the evaluation of multiple integrals for multiloop Feynman massless diagrams. A commutative family of graph building operators Hα for ladder diagrams is constructed and investigated. The complete set of eigenfunctions and the corresponding eigenvalues for the operators Hα are found. This enables us to explicitly express a wide class of four-point ladder diagrams and a general two-loop propagator-type master diagram (with arbitrary indices on the lines) as Mellin-Barnes-type integrals. Special cases of these integrals are explicitly evaluated. A certain class of zig-zag four-point and two-point planar Feynman diagrams (relevant to the bi-scalar D-dimensional “fishnet” field theory and to the calculation of the β-function in ϕ4-theory) is considered. The graph building operators and convenient integral representations for these Feynman diagrams are obtained. The explicit form of the eigenfunctions for the graph building operators of the zig-zag diagrams is fixed by conformal symmetry and these eigenfunctions coincide with the 3-point correlation functions in D-dimensional conformal field theories. By means of this approach, we exactly evaluate the diagrams of the zig-zag series in special cases. In particular, we find a fairly simple derivation of the values for the zig-zag multi-loop two-point diagrams for D = 4. The role of conformal symmetry in this approach, especially a connection of the considered graph building operators with conformal invariant solutions of the Yang-Baxter equation is investigated in detail.

Crystal structures and solution conformations of HtrA from Helicobacter pylori reveal pH-dependent oligomeric conversion and conformational rearrangements

Helicobacter pylori is a Gram-negative microaerophilic bacterium that infects over 50 % of the world's population, making it a major risk factor for chronic gastritis, ulcer diseases of the stomach and duodenum, MALT lymphoma, and gastric cancer. The clinical consequences of H. pylori infection are closely linked with the expression of virulence factors secreted by the bacterium. One such virulence factor is high temperature requirement A (HtrA), which possesses chaperone and serine protease activity. In the host stomach, HtrA secreted from H. pylori (HpHtrA) disrupts intercellular adhesions by cleaving epithelial adhesion proteins including E-cadherin and desmoglein-2. This disruption causes intercellular junctions to open, allowing the bacterium to pass through the epithelial barrier, access the intercellular space, and colonize the gastric mucosa. HtrA proteases are well known for their structural complexity, reflected in their diverse oligomer forms and multi-tasking activities in both prokaryotes and eukaryotes. In this study, we determined crystal structures and solution conformations of HpHtrA monomer and trimer, which revealed large domain rearrangements between them. Notably, this is the first report of a monomeric structure in the HtrA family. We further found a pH-dependent dynamic trimer-to-monomer conversion and concurrent conformational changes that seem closely linked with a pH-sensing ability through the protonation of certain Asp residues. These results advance our understanding of the functional roles and the related mechanisms of this protease in bacterial infection, which may shed light on the development of HtrA-targeted therapies for H. pylori-associated diseases.

Nonaxisymmetrical 9,10-diarylanthracenes: Separable swivel conformers and promising optoelectronic properties

The chemical and physical functions of organic molecules are closely related to their conformations in both solutions and solid states, but the successful separation of organic conformers is usually very difficult because of the rapid internal rotation of single bonds. In the current work, we first demonstrate two stable and separable conformers with swivel cross-conjugated configuration based on 9,10-diphenylanthracene derivatives, and they are confirmed by 1H NMR spectra and single crystal analysis. The introduction of two bulky groups at the ortho positions of the two phenyls can enhance the internal rotation barrier of phenyl–anthryl single bond to fully stabilize the conformers in respective conformation even in reflux toluene. We find that they show similar solution but very different solid-state optical properties such as different emission wavelength and fluorescence efficiency. Moreover, only the syn conformer crystal can exhibit mechanochromic luminescence and mechanoluminescence.

Structural Identification and Antioxidant Activity of Loach Protein Enzymatic Hydrolysates.

Loach, rich in nutrients, such as proteins, amino acids, and mineral elements, is being gradually favored by consumers. Therefore, in this study, the antioxidant activity and structural characteristics of loach peptides were comprehensively analyzed. The loach protein (LAP) with a molecular weight between 150 and 3000 Da was graded by ultrafiltration and nanofiltration processes, which exhibited excellent scavenging activity against DPPH radical (IC50 2.91 ± 0.02 mg/mL), hydroxyl radical (IC50 9.95 ± 0.03 mg/mL), and superoxide anion radical (IC50 13.67 ± 0.33 mg/mL). Additionally, LAP was purified by gel filtration chromatography, and two principal components (named as LAP-I and LAP-II) were isolated. A total of 582 and 672 peptides were identified in LAP-I and LAP-II, respectively, through structural analysis. The XRD results revealed that LAP-I and LAP-II had an irregular amorphous structure. The 2D-NMR spectroscopy results suggested that LAP-I had a compact stretch conformation in the D2O solution, while LAP-II had a folded conformation. Overall, the study results suggested that loach peptide could be a potential antioxidant agent and might provide valuable information for chain conformation and antioxidant mechanism research further.

The role of solute conformation, solvent–solute and solute–solute interactions in crystal nucleation

AbstractThe induction time for nucleation can differ based on the solutions used to conduct a crystallization, which can in turn impact the efficiency and economics of a crystallization process, the crystal size distribution, the morphology and ultimately functionality of the final product. Establishing a link between the nucleation pathway/solution structure and nucleation induction time is essential to achieve improved comprehension of the process of crystal nucleation from solution. In this study, the role of solute conformation, solvent–solute interaction, and solute–solute interaction in nucleation was examined using tolbutamide as a model compound in toluene and toluene–alcohol solutions. Through a combination of induction time experiments, attenuated total reflection Fourier transformed infrared spectroscopy, nuclear magnetic resonance spectroscopy, molecular dynamics simulations, and quantum chemical calculations, it was found that not only solvent–solute interactions but also solute–solute interactions and structural similarities between molecular self‐assemblies in the solution and synthons in the crystal structure, can significantly influence the nucleation induction time.

Stereochemical Analysis of Trifluoroacetamide Derivatives Based on Through-Space 1H–19F Spin–Spin Couplings

In this study, the conformational properties of tertiary trifluoroacetamides in dibenzoazepine (1a and 1b) and benzodiazepine (2a and 2b) derivatives, which exist as equilibrated E- and Z-amide conformers in solution, were investigated by 1H and 19F NMR spectroscopy. In all cases, one of the methylene protons neighboring the nitrogen atom of the minor conformer showed a finely split pattern due to coupling with the trifluoromethyl fluorine atoms, as confirmed by 19F-decoupling experiments. One-dimensional (1D) and two-dimensional (2D) 1H-19F heteronuclear Overhauser spectroscopy (HOESY) experiments were performed to confirm whether these couplings are attributable to through-bond spin-spin couplings (TBCs) or through-space spin-spin couplings (TSCs). HOESY cross-peaks between CF3 (19F) and one of the CH2-N protons of the minor conformers indicate that the two nuclei are spatially close to each other, thus establishing the stereochemistry of the major (E-) and minor (Z-) conformers. The E-amide preferences of the trifluoroacetamides are consistent with the results of density functional theory calculations and X-ray crystallographic analyses. Furthermore, the otherwise incomprehensible 1H NMR spectra were accurately assigned using the HOESY-determined TSCs. The 1H NMR assignments of the E- and Z-methyl signals of N,N-dimethyl trifluoroacetamide, the simplest tertiary trifluoroacetamide, were revised for the first time in half a century.

Ensemble determination by NMR data deconvolution.

Nuclear magnetic resonance (NMR)is the spectroscopic technique of choice for determining molecular conformations in solution at atomic resolution. As solution NMR spectra are rich in structural and dynamic information, the way in whichthe data should be acquired and handled to deliver accurate ensembles is not trivial. This Review provides a guide to the NMRexperiment selection and parametrization process, the generation of viable theoretical conformer pools and the deconvolution of time-averaged NMR data into a conformer ensemble that accurately represents a flexible molecule in solution. In addition to reviewing the key elements of solution ensemble determination of flexible mid-sized molecules, the feasibility and pitfalls of data deconvolution are discussed with a comparison of the performance of representative algorithms.

The intrinsically disordered protein glue of the myelin major dense line: Linking AlphaFold2 predictions to experimental data

Numerous human proteins are classified as intrinsically disordered proteins (IDPs). Due to their physicochemical properties, high-resolution structural information about IDPs is generally lacking. On the other hand, IDPs are known to adopt local ordered structures upon interactions with e.g. other proteins or lipid membrane surfaces. While recent developments in protein structure prediction have been revolutionary, their impact on IDP research at high resolution remains limited. We took a specific example of two myelin-specific IDPs, the myelin basic protein (MBP) and the cytoplasmic domain of myelin protein zero (P0ct). Both of these IDPs are crucial for normal nervous system development and function, and while they are disordered in solution, upon membrane binding, they partially fold into helices, being embedded into the lipid membrane. We carried out AlphaFold2 predictions of both proteins and analysed the models in light of experimental data related to protein structure and molecular interactions. We observe that the predicted models have helical segments that closely correspond to the membrane-binding sites on both proteins. We furthermore analyse the fits of the models to synchrotron-based X-ray scattering and circular dichroism data from the same IDPs. The models are likely to represent the membrane-bound state of both MBP and P0ct, rather than the conformation in solution. Artificial intelligence-based models of IDPs appear to provide information on the ligand-bound state of these proteins, instead of the conformers dominating free in solution. We further discuss the implications of the predictions for mammalian nervous system myelination and their relevance to understanding disease aspects of these IDPs.

Diffuse-Interface Blended Method for Imposing Physical Boundaries in Two-Fluid Flows.

Multiphase flows are commonly found in chemical engineering processes such as distillation columns, bubble columns, fluidized beds and heat exchangers. The physical boundaries of domains in numerical simulations of multiphase flows are generally defined by a conformal unstructured mesh which, depending on the complexity of the physical system, results in time-consuming mesh generation which frequently requires user-intervention. Furthermore, the resulting conformal unstructured mesh could potentially contain a large number of skewed elements, which is undesirable for numerical stability and accuracy. The diffuse-interface approach allows for the use of a simple structured meshes to be used while still capturing the desired physical (e.g., solid-fluid) boundaries. In this work, a novel diffuse-interface method for the imposition of physical boundaries is developed for the incompressible two-fluid multiphase flow model. This model is appropriate for dispersed multiphase flows which are pervasive in chemical engineering processes, in that this flow regime results in high levels of mass and energy transfer between phases. A diffuse interface is used to define the physical boundaries and boundary conditions are imposed by blending the conservation equations from the two-fluid model with that of the nondeformable solid. The results from the diffuse-interface method are compared with results from a conformal unstructured mesh for different interface functions and widths. For small interface widths, the accuracy of the flow profile is unaffected by the choice of interface function and the phase fraction distribution and flow behavior are within 3% compared to those from a conformal mesh. As the interface width increases, the diffuse-interface solution deviates from the conformal mesh solution in both the localized gas fraction and the overall gas hold-up, resulting in a difference up to 30%. In the case of flow past a cylinder, where the solid interacts with the flow, the presence of the diffuse interface extends the thickness of the solid boundary and results in a deviation from the conformal mesh solution as time increases.

Topology-Directed Self-Locking of Colloidal Suprastructures

The robustness and stability of self-assembled colloidal suprastructures are pivotal and challenging in the field of soft condensed matter and nanotechnology. Herein, we develop a novel approach to “self-locking” the colloidal suprastructures to achieve excellent stability by taking advantage of the topology of polymer brushes on nanoparticles. Compared to linear polymer brushes, looped polymer brushes can significantly improve the structural stability of the suprastructures. A combination of experiments and simulations reveals that the suprastructures achieve their significant stabilities through a “self-locking” mechanism by forming folded entanglement between looped polymer molecules. The stabilized suprastructures can stay intact in various organic solvent environments or under heating and mechanical oscillations. Furthermore, the interparticle spacing of suprastructures can be modulated by controlling polymer conformations in an aqueous solution through varying salt concentrations. This new approach of “self-locking” provides an easy-to-operate strategy to stabilize suprastructures and modulate interparticle distances without adding chemicals, which allows the potentially wide applications of colloidal suprastructures under harsh environments.