Conformational Differences Research Articles

TMEM63 proteins function as monomeric high-threshold mechanosensitive ion channels

OSCA/TMEM63s form mechanically activated (MA) ion channels in plants and animals, respectively. OSCAs and related TMEM16s and transmembrane channel-like (TMC) proteins form homodimers with two pores. Here, we uncover an unanticipated monomeric configuration of TMEM63 proteins. Structures of TMEM63A and TMEM63B (referred to as TMEM63s) revealed a single highly restricted pore. Functional analyses demonstrated that TMEM63s are bona fide mechanosensitive ion channels, characterized by small conductance and high thresholds. TMEM63s possess evolutionary variations in the intracellular linker IL2, which mediates dimerization in OSCAs. Replacement of OSCA1.2 IL2 with TMEM63A IL2 or mutations to key variable residues resulted in monomeric OSCA1.2 and MA currents with significantly higher thresholds. Structural analyses revealed substantial conformational differences in the mechano-sensing domain IL2 and gating helix TM6 between TMEM63s and OSCA1.2. Our studies reveal that mechanosensitivity in OSCA/TMEM63 channels is affected by oligomerization and suggest gating mechanisms that may be shared by OSCA/TMEM63, TMEM16, and TMC channels.

Mechanistic elucidation of the degradation and transformation of hydroxy-α-sanshool and its conformers as the pungent dietary components in Sichuan pepper: A DFT study

To better understand the structural changes of sanshool pungent dietary components during the process and preservation of Sichuan pepper and pungent foods, the mechanistic insights into the intrinsic degradation and transformation of 16 hydroxy-α-sanshool conformers have been explored computationally. Our results have revealed that increasing the cis-CC bond numbers in the most stable all-trans hydroxy-β-sanshool structure causes the maximum 34.21 kJ/mol conformational energetic difference, and the existent probability of C2nnn would be lower than that of C1nnn (n = 1,2). The isomerization between the conformers could be much easier when they are excited by light radiation, as the strength of the CC bonds and their connected CC bonds becomes significantly close, and the relative energies among conformers are largely reduced. Besides, the different combination of cis/trans-CC bonds changes the delocalization degree of molecular Frontier orbitals, which consequently causes the different photochemical stability. Finally, the possible molecular oxidation degradation mechanism is proposed.

Explaining Differences in Conformity between Republican CEOs and Democratic CEOs

Explaining Differences in Conformity between Republican CEOs and Democratic CEOs

Monitoring the macrophage response towards biomaterial implants using label-free imaging.

Understanding the immune system's foreign body response (FBR) is essential when developing and validating a biomaterial. Macrophage activation and proliferation are critical events in FBR that can determine the material's biocompatibility and fate in vivo. In this study, two different macro-encapsulation pouches intended for pancreatic islet transplantation were implanted into streptozotocin-induced diabetes rat models for 15 days. Post-explantation, the fibrotic capsules were analyzed by standard immunohistochemistry as well as non-invasive Raman microspectroscopy to determine the degree of FBR induced by both materials. The potential of Raman microspectroscopy to discern different processes of FBR was investigated and it was shown that Raman microspectroscopy is capable of targeting ECM components of the fibrotic capsule as well as pro and anti-inflammatory macrophage activation states, in a molecular-sensitive and marker-independent manner. In combination with multivariate analysis, spectral shifts reflecting conformational differences in Col I were identified and allowed to discriminate fibrotic and native interstitial connective tissue fibers. Moreover, spectral signatures retrieved from nuclei demonstrated changes in methylation states of nucleic acids in M1 and M2 phenotypes, relevant as indicator for fibrosis progression. This study could successfully implement Raman microspectroscopy as complementary tool to study in vivo immune-compatibility providing insightful information of FBR of biomaterials and medical devices, post-implantation.

Diverse p120RasGAP interactions with doubly phosphorylated partners EphB4, p190RhoGAP, and Dok1

RasGAP (p120RasGAP), the founding member of the GTPase-activating protein (GAP) family, is one of only nine human proteins to contain two SH2 domains and is essential for proper vascular development. Despite its importance, its interactions with key binding partners remains unclear. In this study we provide a detailed viewpoint of RasGAP recruitment to various binding partners and assess their impact on RasGAP activity. We reveal the RasGAP SH2 domains generate distinct binding interactions with three well-known doubly phosphorylated binding partners: p190RhoGAP, Dok1, and EphB4. Affinity measurements demonstrate a 100-fold weakened affinity for RasGAP-EphB4 binding compared to RasGAP-p190RhoGAP or RasGAP-Dok1 binding, possibly driven by single versus dual SH2 domain engagement with a dominant N-terminal SH2 interaction. Small-angle X-ray scattering reveals conformational differences between RasGAP-EphB4 binding and RasGAP-p190RhoGAP binding. Importantly, these interactions do not impact catalytic activity, implying RasGAP utilizes its SH2 domains to achieve diverse spatial-temporal regulation of Ras signaling in a previously unrecognized fashion.

Comparative Biochemical, Structural, and Functional Analysis of Recombinant Phospholipases D from Three Loxosceles Spider Venoms.

Spiders of Loxosceles genus are widely distributed and their venoms contain phospholipases D (PLDs), which degrade phospholipids and trigger inflammatory responses, dermonecrosis, hematological changes, and renal injuries. Biochemical, functional, and structural properties of three recombinant PLDs from L. intermedia, L. laeta, and L. gaucho, the principal species clinically relevant in South America, were analyzed. Sera against L. gaucho and L. laeta PLDs strongly cross-reacted with other PLDs, but sera against L. intermedia PLD mostly reacted with homologous molecules, suggesting underlying structural and functional differences. PLDs presented a similar secondary structure profile but distinct melting temperatures. Different methods demonstrated that all PLDs cleave sphingomyelin and lysophosphatidylcholine, but L. gaucho and L. laeta PLDs excelled. L. gaucho PLD showed greater "in vitro" hemolytic activity. L. gaucho and L. laeta PLDs were more lethal in assays with mice and crickets. Molecular dynamics simulations correlated their biochemical activities with differences in sequences and conformations of specific surface loops, which play roles in protein stability and in modulating interactions with the membrane. Despite the high similarity, PLDs from L. gaucho and L. laeta venoms are more active than L. intermedia PLD, requiring special attention from physicians when these two species prevail in endemic regions.

Structural insights into redox signal transduction mechanisms in the control of nitrogen fixation by the NifLA system

NifL is a conformationally dynamic flavoprotein responsible for regulating the activity of the σ54-dependent activator NifA to control the transcription of nitrogen fixation (nif) genes in response to intracellular oxygen, cellular energy, or nitrogen availability. The NifL-NifA two-component system is the master regulatory system for nitrogen fixation. NifL serves as a sensory protein, undergoing signal-dependent conformational changes that modulate its interaction with NifA, forming the NifL-NifA complex, which inhibits NifA activity in conditions unsuitable for nitrogen fixation. While NifL-NifA regulation is well understood, these conformationally flexible proteins have eluded previous attempts at structure determination. In work described here, we advance a structural model of the NifL dimer supported by a combination of scattering techniques and mass spectrometry (MS)-coupled structural analyses that report on the average structure in solution. Using a combination of small angle X-ray scattering-derived electron density maps and MS-coupled surface labeling, we investigate the conformational dynamics responsible for NifL oxygen and energy responses. Our results reveal conformational differences in the structure of NifL under reduced and oxidized conditions that provide the basis for a model for modulating NifLA complex formation in the regulation of nitrogen fixation in response to oxygen in the model diazotroph, Azotobacter vinelandii.

Protective mechanism of quercetin compounds against acrylamide-induced hepatotoxicity

Quercetin compounds have antioxidant, anti-inflammatory and anticancer pharmacological functions. Long-term exposure to acrylamide (AA) can cause liver injury and endanger human health. However, whether quercetin compounds can attenuate AA-induced liver injury and the specific mechanism are not clear. Here, we studied the mechanism and structure-activity relationship of quercetin compounds in reducing AA-induced hepatotoxicity in vivo and in vitro. In vivo studies found that quercetin-like compounds protect against AA-induced liver injury by reducing oxidative stress levels, activating the Akt/mTOR signaling pathway to attenuate autophagy, and improving mitochondrial apoptosis and endoplasmic reticulum stress-mediated apoptosis. In vitro studies found that quercetin compounds protected HepG2 cells from AA by attenuating the activation of AA-induced autophagy, lowering reactive oxygen species (ROS) levels by exerting antioxidant effects and thus attenuating oxidative stress, increasing mitochondrial membrane potential (MMP), and improving apoptosis-related proteins, thus attenuating AA-induced apoptosis. Furthermore, the conformational differences between quercetin compounds correlated with their protective capacity against AA-induced hepatotoxicity, with quercetin showing the best protective capacity due to its strongest antioxidant activity. In conclusion, quercetin compounds can protect against AA-induced liver injury through multiple pathways of oxidative stress, autophagy and apoptosis, and their protective capacity correlates with antioxidant activity.

Structural and Computational Analyses of the Unique Interactions of Opicapone in the Binding Pocket of Catechol O-Methyltransferase: A Crystallographic Study and Fragment Molecular Orbital Analyses.

A third-generation inhibitor of catechol O-methyltransferase (COMT), opicapone (1), has the 3-nitrocatechol scaffold as do the second-generation inhibitors such as entacapone (2) and tolcapone (3), but only 1 can sustainably inhibit COMT activity making it suitable for a once-daily regimen. These improvements should be attributed to the optimized sidechain moiety (oxidopyridyloxadiazolyl group) of 1 substituted at the 5-position of the 3-nitrocatechol ring. We analyzed the role of the sidechain moiety by solving the crystal structures of COMT/S-adenosylmethionine (SAM)/Mg/1 and COMT/S-adenosylhomocysteine (SAH)/Mg/1 complexes. Fragment molecular orbital (FMO) calculations elucidated that the dispersion interaction between the sidechains of Leu 198 and Met 201 on the β6β7-loop and the oxidopyridine ring of 1 were unique and important in both complexes. In contrast, the catechol binding site made a remarkable difference in the sidechain conformation of Lys 144. The ε-amino group of Lys 144 was outside of the catalytic pocket and was replaced by a water molecule in the COMT/SAH/Mg/1 complex. No nitrocatechol inhibitor has ever been reported to make a complex with COMT and SAH. Thus, the conformational change of Lys 144 found in the COMT/SAH/Mg/1 complex is the first crystallographic evidence that supports the role of Lys 144 as a catalytic base to take out a proton ion from the reaction site to the outside of the enzyme. The fact that 1 generated a complex with SAH and COMT also suggests that 1 could inhibit COMT twofold, as a typical substrate mimic competitive inhibitor and as a product-inhibition enhancer.

Degradation and Defluorination of Per- and Polyfluoroalkyl Substances by Direct Photolysis at 222 nm.

The susceptibility of 19 representative per- and polyfluoroalkyl substances (PFAS) to direct photolysis and defluorination under far-UVC 222 nm irradiation was investigated. Enhanced photolysis occurred for perfluorocarboxylic acids (PFCAs), fluorotelomer unsaturated carboxylic acids (FTUCAs), and GenX, compared to that at conventional 254 nm irradiation on a similar fluence basis, while other PFAS showed minimal decay. For degradable PFAS, up to 81% of parent compound decay (photolysis rate constant (k222nm) = 8.19-34.76 L·Einstein-1; quantum yield (Φ222nm) = 0.031-0.158) and up to 31% of defluorination were achieved within 4 h, and the major transformation products were shorter-chain PFCAs. Solution pH, dissolved oxygen, carbonate, phosphate, chloride, and humic acids had mild impacts, while nitrate significantly affected PFAS photolysis/defluorination at 222 nm. Decarboxylation is a crucial step of photolytic decay. The slower degradation of short-chain PFCAs than long-chain ones is related to molar absorptivity and may also be influenced by chain-length dependent structural factors, such as differences in pKa, conformation, and perfluoroalkyl radical stability. Meanwhile, theoretical calculations indicated that the widely proposed HF elimination from the alcohol intermediate (CnF2n+1OH) of PFCA is an unlikely degradation pathway due to high activation barriers. These new findings are useful for further development of far-UVC technology for PFAS in water treatment.

Binding modes of potential anti-prion phytochemicals to PrPC structures in silico

BackgroundPrion diseases involve the conversion of a normal, cell-surface glycoprotein (PrPC) into a misfolded pathogenic form (PrPSc). One possible strategy to inhibit PrPSc formation is to stabilize the native conformation of PrPC and interfere with the conversion of PrPC to PrPSc. Many compounds have been shown to inhibit the conversion process, however, no promising drugs have been identified to cure prion diseases. ObjectiveThis study aims to identify potential anti-prion compounds from plant phytochemicals by integrating traditional ethnobotanical knowledge with modern in silico drug design approaches. Materials and methodsIn the current study medicinal phytochemicals were docked with swapped and non-swapped crystal structures of PrPCin silico to identify potential anti-prions to determine their binding modes and interactions. ResultsEleven new phytochemicals were identified based on their binding energies and pharmacokinetic properties. The binding sites and interactions of the known and new anti-prion compounds are similar, and differences in binding modes occur in structures with very subtle differences in side chain conformations. Binding of these compounds poses steric hindrance to neighbouring molecules. Residues shown to be associated with the inhibition of PrPC to PrPSc conversion form interactions with most of the compounds. ConclusionIdentified compounds might act as potent inhibitors of PrPC to PrPSc conversion. These might be attractive candidates for the development of novel anti-prion therapy although further tests in vitro cell cultures and in vivo mouse models are needed to confirm these findings.

Peer Conformity in Terms of Gender

Peer conformity is one of the causes of problems that many students do at school. This study aims to describe peer conformity and differences in peer conformity in terms of gender. This research uses a quantitative method with a comparative descriptive research type. The population in this study were 631 students in grades X, XI, and XII SMA Adabiah Padang for the academic year 2022/2023 and a sample of 259 students was drawn using proportional random sampling. Collecting data in this study using a peer conformity questionnaire. Data were processed using descriptive analysis and t-test with the help of SPSS version 20. The results showed that peer conformity behavior at Adabiah Padang High School was in the "medium" category. There is a significant difference between students' peer conformity in terms of gender. Percentage of conformity among male students (57.9%) and female students (64.6%) with a significance value (2-tailed) of 0.000. The results of this study have implications for Guidance and Counseling service activities.

Preparation and Characterization of Carbazole-Based Luminogen with Efficient Emission in Solid and Solution States.

Organic luminescent materials with high luminescence efficiency in both solution and solid states, namely dual-state emission (DSE), have attracted considerable attention due to their promising applications in various fields. In order to enrich the variety of DSE materials, carbazole, similar to triphenylamine (TPA), was utilized to construct a novel DSE luminogen named 2-(4-(9H-carbazol-9-yl)phenyl)benzo[d]thiazole (CZ-BT). CZ-BT exhibited DSE characteristics with fluorescence quantum yields of 70, 38 and 75% in solution, amorphous and crystalline states, respectively. CZ-BT shows thermochromic and mechanochromic properties in solution and solids, respectively. Theoretical calculations show that there is a small conformational difference between the ground state and the lowest singly excited state of CZ-BT and that it exhibits a low non-radiative transition characteristic. The oscillator strength during the transition from the single excited state to the ground state reaches 1.0442. CZ-BT adopts a distorted molecular conformation with intramolecular hindrance effects. The excellent DSE properties of CZ-BT can be explained well using theoretical calculations and experimental results. In terms of application, the CZ-BT has a detection limit for the hazardous substance picric acid of 2.81 × 10-7 mol/L.

Computational insights into novel inhibitor indole-heterocycle specific against glycogen phosphorylase isoenzymes interaction mechanism.

Background: Glycogen phosphorylase (GP) is a potential drug target. As the three subtypes of GP are highly conserved, it is difficult to research their specificity. However, compound 1 inhibits the GP subtypes differently and was studied to aid in designing specific inhibitors. Results: Molecular docking showed that the ligands in GP subtype complexes had some differences in spatial conformation and binding modes, stabilized by polar and nonpolar interactions. The results were confirmed through kinetic experiments, with affinities of -85.230 (brain GP), -73.809 (liver GP) and -66.061kJ/mol (muscle GP). Conclusion: The study provides insight into the possible reasons for differences in compound 1's inhibitory activity against the GP subtypes and offers guidance in designing target molecules for regulating selectivity among the subtypes.

Morphological characteristics and genetic diversity of Terapon jarbua (Forrskäl, 1775) in Central, Vietnam

Many environmental factors affect the morphology of migratory fish species, such as salinity, water flow rate, and temperature. However, studies on changes in fish morphology under environmental variations from salt water to brackish water are still limited in many fish species, especially in Terapon jarbua. This study aims to investigate the differences in the morphological parameters of T. jarbua between the coastal sea (seawater) and lagoon (brackish water); and between male and female fish based on a landmark morphological approach. Additionally, the genetic diversity of T. jarbua populations in Central Vietnam was elucidated using the mitochondrial DNA cytochrome oxidase subunit I (mtDNA COI) sequence as a molecular marker. The analytical results indicated no sexual dimorphism in the T. jarbua population, yet conformational differences exist between the two studied aquatic species. The analysis of 42 mtDNA COI sequences collected from Central Vietnam identified 13 haplotypes with medium genetic diversity and low genetic differentiation between the Tam Giang lagoon and Thua Thien Hue coastal (Fst = 0.028) and not significant (p = 0.126). Most haplotypes obtained are present in reference populations, indicating a high genetic exchange between populations. We proposed that the T. jarbua population in Central Vietnam has a stable connection with neighboring populations (China, Taiwan, Philippines, Indonesia, Malaysia, Bangladesh, India, and Pakistan).

MOF-derived magnetic trimetallic oxide-carbon nanocomposites for modification-free and dual molecular events-dependent ratiometric electrochemical analysis of intracellular glutathione

The response of ratiometric electrochemical signals toward the targets of interest generally depends on specific molecules-mediated single molecular recognition event, which limits the application in analyzing the targets without specific recognition molecules. In this work, novel dual molecular recognition events were proposed for rapid and reliable electrochemical analysis of glutathione (GSH) using MOF-derived trimetallic oxide-carbon (Fe3O4/CuO/NiO-C) nanocomposites. The binding of GSH to CuO caused a decrease in the oxidation signal of Cu(Ⅱ) due to the formation of nonelectroactive complex, while NiO could electrocatalyze the oxidation of GSH. These GSH-involved dual molecular recognition events guaranteed the readout of ratiometric electrochemical signals with a complementary response range, allowing sensitive and wide-range analysis of GSH. Besides, the difference in spatial conformation gave rise to a different oxidation potential for different biothiols, which guaranteed selective analysis of GSH. The presence of Fe3O4 allowed modification-free detection within 5 min using a magnetic working electrode. Taking advantage of these merits, the developed electrochemical sensor could rapidly and reliably monitor the intracellular GSH.

Structural assessment of the full-length wild-type tumor suppressor protein p53 by mass spectrometry-guided computational modeling

The tetrameric tumor suppressor p53 represents a great challenge for 3D-structural analysis due to its high degree of intrinsic disorder (ca. 40%). We aim to shed light on the structural and functional roles of p53’s C-terminal region in full-length, wild-type human p53 tetramer and their importance for DNA binding. For this, we employed complementary techniques of structural mass spectrometry (MS) in an integrated approach with computational modeling. Our results show no major conformational differences in p53 between DNA-bound and DNA-free states, but reveal a substantial compaction of p53’s C-terminal region. This supports the proposed mechanism of unspecific DNA binding to the C-terminal region of p53 prior to transcription initiation by specific DNA binding to the core domain of p53. The synergies between complementary structural MS techniques and computational modeling as pursued in our integrative approach is envisioned to serve as general strategy for studying intrinsically disordered proteins (IDPs) and intrinsically disordered region (IDRs).

The impact of modified polystyrene on lysozyme fibrillation studied by surface-enhanced Raman spectroscopy (SERS)

Nanoplastics could modulate the fibrillation of amyloid proteins. However, many chemical functional groups are adsorbed to change the interfacial chemistry of nanoplastics in the real world. Herein, this study aimed to investigate the effects of polystyrene (PS), carboxyl modified PS (PS-COOH), and amino modified PS (PS-NH2) on the fibrillation of hen egg-white lysozyme (HEWL). Due to the differences in the interfacial chemistry, concentration was considered an essential factor. PS-NH2 (10 μg/mL) could promote the fibrillation of HEWL similar to PS (50 μg/mL) and PS-COOH (50 μg/mL). Moreover, promoting the primary nucleation step of amyloid fibril formation was the primary reason. The differences in spatial conformation of HEWL were characterized by Fourier transform-infrared spectroscopy and surface enhanced Raman spectroscopy (SERS). Strikingly, a particular signal of SERS of HEWL incubated with PS-NH2 at 1610 cm−1 was found due to the interaction between amino group of PS-NH2 and tryptophan (or tyrosine) of HEWL. Therefore, a new perspective was provided to understand the regulation of interfacial chemistry of nanoplastics on the fibrillation of amyloid proteins. Additionally, this study suggested that SERS could be a powerful method to investigate the interactions between proteins and nanoparticles.

Casein micelle with different β-casein phenotypes: Fingerprinting pH-induced structural changes using FTIR and NMR spectroscopies

The aim of this study was to investigate the structural characteristics of casein micelles and casein particles at pH 6.7, 5.7, and 2.3 containing β-caseins A1/A1, A1/A2, and A2/A2 at 37 °C using Nuclear Magnetic Resonance, Fourier-Transform Infrared spectroscopies and chemometrics. The amount of all released caseins from the casein micelle at pH 6.7 was significantly different after the pH had been reduced to 5.7 and subsequently decreased to 2.3. Results showed variation in the structure of all three samples mainly dependent of caseins content, caseins’ phenotypes, and pH modulation. During the pH modulation, higher levels of α-helixes and intramolecular β-sheets were found in A1/A1 casein, whilst aggregated β-sheets, β-turns, and polyproline II helixes dominated in A1/A2 and A2/A2 samples. Principal Component Analysis was used to characterise and distinguish among the structure of the three caseins based on spectral data. While samples containing β-casein A2 possessed structural properties different to A1/A1 samples at pH 6.7 and 2.3, at pH 5.7 all casein micelles behaved in a similar manner. This study brings significant insights in the importance of the observed casein phenotypes on the structural arrangement of A1/A1, A1/A2, and A2/A2 casein micelles, hence defining their main conformational differences essential for the dairy industry.

Conformational Distributions of Phenyl β-D-Glucopyranoside and Gastrodin in Solution by Vibrational Optical Activity and Theoretical Calculations.

The conformational landscapes of two highly flexible monosaccharide derivatives, namely phenyl β-D-glucopyranoside (ph-β-glu) and 4-(hydroxymethyl)phenyl β-D-glucopyranoside, also commonly known as gastrodin, were explored using a combined experimental and theoretical approach. For the infrared, Raman, and the associated vibrational optical activity (VOA), i.e., vibrational circular dichroism and Raman optical activity, experiments of these two compounds in DMSO and in water were carried out. Extensive and systematic conformational searches were performed using a recently developed conformational searching tool called CREST (conformer-rotamer ensemble sampling tool) in the two solvents. Fourteen and twenty-four low-energy conformers were identified at the DFT level for ph-β-glu and gastrodin, respectively. The spectral simulations of individual conformers were done at the B3LYP-D3BJ/def2-TZVPD level with the polarizable continuum model of the solvents. The VOA spectral features exhibit much higher specificity to conformational differences than their parent infrared and Raman. The excellent agreements achieved between the experimental and simulated VOA spectra allow for the extraction of experimental conformational distributions of these two carbohydrates in solution directly. The experimental percentage abundances based on the hydroxymethyl (at the pyranose ring) conformations G+, G-, and T for ph-β-glu were obtained to be 15%, 75%, and 10% in DMSO and 53%, 40%, and 7% in water, respectively, in comparison to the previously reported gas phase values of 68%, 25%, and 7%, highlighting the important role of solvents in conformational preferences. The corresponding experimental distributions for gastrodin are 56%, 22%, and 22% in DMSO and 70%, 21%, and 9% in water.