Conformational Changes Research Articles

Enhanced sampling of protein conformational changes via true reaction coordinates from energy relaxation

The bottleneck in enhanced sampling lies in finding collective variables that effectively accelerate protein conformational changes; true reaction coordinates that accurately predict the committor are the well-recognized optimal choice. However, identifying them requires unbiased natural reactive trajectories, which, paradoxically, require effective enhanced sampling. Using the generalized work functional method, we uncover that true reaction coordinates control both conformational changes and energy relaxation, enabling us to compute them from energy relaxation simulations. Biasing true reaction coordinates accelerates conformational changes and ligand dissociation in PDZ2 domain and HIV-1 protease by 105 to 1015-fold. The resulting trajectories follow natural transition pathways, enabling efficient generation of unbiased reactive trajectories. In contrast, biased trajectories from empirical collective variables display non-physical features. Furthermore, our method uses a single protein structure as input, enabling predictive sampling of conformational changes. These findings unlock access to a broader range of protein functional processes in molecular dynamics simulations.

Molybdate uptake interplay with ROS tolerance modulates bacterial pathogenesis.

The rare metal element molybdenum functions as a cofactor in molybdoenzymes that are essential to life in almost all living things. Molybdate can be captured by the periplasmic substrate-binding protein ModA of ModABC transport system in bacteria. We demonstrate that ModA plays crucial roles in growth, multiple metabolic pathways, and ROS tolerance in Acinetobacter baumannii. Crystal structures of molybdate-coordinated A. baumannii ModA show a noncanonical disulfide bond with a conformational change between reduced and oxidized states. Disulfide bond formation reduced binding affinity to molybdate by two orders of magnitude and contributes to its substrate preference. ModA-mediated molybdate binding was important for A. baumannii infection in a murine pneumonia model. Together, our study sheds light on the structural and functional diversity of molybdate uptake and highlights a potential target for antibacterial development.

Role of Copper and Zinc Ions in the Hydrolytic Degradation of Neurodegeneration-Related Peptides

Spontaneous cleavage reactions normally occur in vivo on amino acid peptide backbones, leading to fragmentation products that can have different physiological roles and toxicity, particularly when the substrate of the hydrolytic processes are neuronal peptides and proteins highly related to neurodegeneration. We report a hydrolytic study performed with the HPLC-MS technique at different temperatures (4 °C and 37 °C) on peptide fragments of different neuronal proteins (amyloid-β, tau, and α-synuclein) in physiological conditions in the presence of Cu2+ and Zn2+ ions, two metal ions found at millimolar concentrations in amyloid plaques. The coordination of these metal ions with these peptides significantly protects their backbones toward hydrolytic degradation, preserving the entire sequences over two weeks in solution, while the free peptides in the same buffer are fully fragmented after the same or even shorter incubation period. Our data show that peptide cleavage is not only ruled by the chemical sensitivity of amino acids, but the peptide conformation changes induced by metal coordination influence hydrolytic reactions. The enhanced stability of neuronal peptides provided by metal coordination can increase local levels of amyloidogenic species capable of seeding fibril growth, resulting in aberrant protein depositions and deficits in neuronal activity.

Structure of the Kaposi's sarcoma-associated herpesvirus gB in post-fusion conformation.

Discovered in 1994 in lesions of an AIDS patient, Kaposi's sarcoma-associated herpesvirus (KSHV) is a member of the gammaherpesvirus subfamily of the Herpesviridae family, which contains a total of nine that infect humans. These viruses all contain a large envelope glycoprotein, glycoprotein B (gB), that is required for viral fusion with host cell membrane to initial infection. Although the atomic structures of five other human herpesviruses in their postfusion conformation and one in its prefusion conformation are known, the atomic structure of KSHV gB has not been reported. Here, we report the first structure of the KSHV gB ectodomain determined by single-particle cryogenic electron microscopy (cryoEM). Despite a similar global fold between herpesvirus gB, KSHV gB possesses local differences not shared by its relatives in other herpesviruses. The glycosylation sites of gB are arranged in belts down the symmetry axis with distinct localization compared to that of other herpesviruses, which occludes certain antibody binding sites. An extended glycan chain observed in domain I (DI), located proximal to the host membrane, may suggest its possible role in host cell attachment. Local flexibility of domain IV (DIV) governed by molecular hinges at its interdomain junctions identifies a means for enabling conformational change. A mutation in the domain III (DIII) central helix disrupts incorporation of gB into KSHV virions despite adoption of a canonical fold in vitro. Taken together, this study reveals mechanisms of structural variability of herpesvirus fusion protein gB and informs its folding and immunogenicity.IMPORTANCEIn 1994, a cancer-causing virus was discovered in lesions of AIDS patients, which was later named Kaposi's sarcoma-associated herpesvirus (KSHV). As the latest discovered human herpesvirus, KSHV has been classified into the gammaherpesvirus subfamily of the Herpesviridae. In this study, we have expressed KSHV gB and employed cryogenic electron microscopy (cryoEM) to determine its first structure. Importantly, our structure resolves some glycans beyond the first sugar moiety. These glycans are arranged in a pattern unique to KSHV, which impacts the antigenicity of KSHV gB. Our structure also reveals conformational flexibility caused by molecular hinges between domains that provide clues into the mechanism behind the drastic change between prefusion and postfusion states.

FRET analysis of the unwrapping of nucleosomal DNA containing a sequence characteristic of the + 1 nucleosome

Sequence-dependent mechanical properties of DNA could play essential roles in nuclear processes by affecting histone-DNA interactions. Previously, we found that the DNA entry site of the first nucleosomes from the transcription start site (+ 1 nucleosome) in budding yeast enriches AA/TT steps, but not the exit site, and the biased presence of AA/TT in the entry site was associated with the transcription levels of yeast genes. Because AA/TT is a rigid dinucleotide step, we considered that AA/TT causes DNA unwrapping. However, our previous MNase-seq experiments with reconstituted nucleosomes left some doubt regarding this interpretation, owing to its high exonuclease activity. Furthermore, MNase cleavage did not provide direct evidence of its structural state. In this study, Förster resonance energy transfer (FRET) measurements were used to investigate salt-induced conformational changes in nucleosomal DNA containing AA/TT repeats at the entry site. We observed that the AA/TT region wrapped around the histone core was as likely as other DNA sequences at physiological salt concentrations. However, it unwrapped at a lower salt concentration, indicating weaker electrostatic interactions with the histone core. Ethidium-induced nucleosome disruption assay showed that the intercalator had greater access to DNA with AA/TT at the entry site. Taken together, these results suggest that AA/TT at the entry sites induces DNA unwrapping from the histone core on the promoter side, which may promote transcriptional activation in response to the approach of transcription-related proteins.

Molecular Determinants of Optical Modulation in ssDNA-Carbon Nanotube Biosensors.

Most traditional optical biosensors operate through molecular recognition, where ligand binding causes conformational changes that lead to optical perturbations in the emitting motif. Optical sensors developed from single-stranded DNA-functionalized single-walled carbon nanotubes (ssDNA-SWCNTs) have started to make useful contributions to biological research. However, the mechanisms underlying their function have remained poorly understood. In this study, we combine experimental and computational approaches to show that ligand binding alone is not sufficient for optical modulation in this class of synthetic biosensors. Instead, the optical response that occurs after ligand binding is highly dependent on the chemical properties of the ligands, resembling mechanisms seen in activity-based biosensors. Specifically, we show that in ssDNA-SWCNT catecholamine sensors, the optical response correlates positively with the electron density on the aryl motif, even among ligands with similar ligand binding affinities. Importantly, despite the strong correlations with electrochemical properties, we find that catechol oxidation itself is not necessary to drive the sensor optical response. We discuss how these findings could serve as a framework for tuning the performance of existing sensors and guiding the development of new biosensors of this class.

Structural insights into RNA cleavage by PIWI Argonaute.

Argonaute proteins are categorized into AGO and PIWI clades. Across most animal species, AGO-clade proteins are widely expressed in various cell types, andregulate normal gene expression1. By contrast, PIWI-clade proteins predominantly function during gametogenesis to suppress transposons and ensure fertility1,2. Both clades use nucleic acid guides for target recognition by means of base pairing, crucial for initiating target silencing, often through direct cleavage. AGO-clade proteins use a narrow channel to secure a tight guide-target interaction3. By contrast, PIWI proteins feature a wider channel that potentially allows mismatches during pairing, broadening target silencing capability4,5. However, the mechanism of PIWI-mediated target cleavage remains unclear. Here we demonstrate that after target binding, PIWI proteins undergo a conformational change from an 'open' stateto a 'locked' state, facilitating base pairing and enhancing target cleavage efficiency. This transition involves narrowing of the binding channel and repositioning of the PIWI-interacting RNA-target duplex towards the MID-PIWI lobe, establishing extensive contacts for duplex stabilization. During this transition, we also identify an intermediate 'comma-shaped' conformation, which might recruit GTSF1, a known auxiliary protein thatenhances PIWI cleavage activity6. GTSF1 facilitates the transition to the locked state by linking the PIWI domain to the RNA duplex, thereby expediting the conformational change critical for efficient target cleavage. These findings explain the molecular mechanisms underlying PIWI-PIWI-interactingRNA complex function in target RNA cleavage, providing insights into how dynamic conformational changes from PIWI proteins coordinate cofactors to safeguard gametogenesis.

Conformational Dynamics of Mitochondrial Inorganic Pyrophosphatase hPPA2 and Its Changes Caused by Pathogenic Mutations

Inorganic pyrophosphatases, or PPases, are ubiquitous enzymes whose activity is necessary for a large number of biosynthetic reactions. The catalytic function of PPases is dependent on certain conformational changes that have been previously characterized based on the comparison of the crystal structures of various complexes. The current work describes the conformational dynamics of a structural model of human mitochondrial pyrophosphatase hPPA2 using molecular dynamics simulation, all-atom principal component analysis, and coarse-grained normal mode analysis. In addition to the wild-type enzyme, four mutant variants of hPPA2 were characterized that correspond to the natural pathogenic variants causing severe mitochondrial dysfunction and cardio pathologies. As a result, we identified the global type of flexible motion that seems to be shared by other dimeric PPases. This motion is discussed in terms of the allosteric behavior of the protein. Analysis of the observed conformational dynamics revealed the formation of a binding site for anionic ligands in the active site that could be relevant to enzyme catalysis. Based on the comparison of the wild-type and mutant PPases dynamics, we suggest the possible molecular mechanisms of the functional incompetence of hPPA2 caused by mutations. The results of this work allow for deeper insight into the structural basis of PPase function and the possible effects of pathogenic mutations on the protein structure and function.

Dual-protein system composed of soy protein and β-lactoglobulin: effect of transglutaminase-mediated crosslinking on its allergenicity and conformation.

The escalating global prevalence of food allergies has intensified the need for hypoallergenic food products. Transglutaminase (TGase)-mediated crosslinking has garnered significant attention for its potential to reduce the allergenicity of food proteins. This study aimed to investigate the effects of TGase crosslinking on the potential allergenicity and conformational changes in a dual-protein system composed of β-lactoglobulin (β-LG) and soy protein isolate (SPI) at varying mass ratios (10:0, 7:3, 5:5, 3:7 and 0:10 (w/w)). TGase preferentially crosslinked the 7S and 11S subunits of soy protein, rather than β-LG. Crosslinking treatment reduced the allergenic potential of both soy protein and β-LG, with the degree of reduction depending on the protein ratio. The β-LG5:SPI5 and β-LG7:SPI3 mixtures showed the most significant reduction in antibody reactivity towards soy protein and β-LG, respectively. Additionally, TGase-mediated crosslinking significantly reduced the binding capacity of all dual-protein samples to serum immunoglobulin E from allergic patients, compared to the control group (P < 0.05). The allergenicity reduction was accompanied by structural modifications, including a decrease in β-sheet content, an increase in β-turn and random coil structures, enhanced ultraviolet absorption and intrinsic fluorescence, reduced free sulfhydryl levels and altered intermolecular forces. These changes suggest that TGase-induced crosslinking may disrupt or mask allergenic epitopes, thus lowering allergenicity. TGase crosslinking effectively reduced the allergenic potential of the dual-protein system by inducing structural changes. These findings underscore the potential of TGase in developing hypoallergenic dual-protein food products and provide a scientific foundation for future research and application in the food industry. © 2025 Society of Chemical Industry.

Ethosuximide: Subunit- and Gβγ-dependent blocker and reporter of allosteric changes in GIRK channels.

The antiepileptic drug ethosuximide (ETX) suppresses epileptiform activity in a mouse model of GNB1 syndrome, caused by mutations in Gβ1 protein, likely through the inhibition of G-protein gated K+ (GIRK) channels. Here, we investigated the mechanism of ETX inhibition (block) of different GIRKs. We studied ETX inhibition of GIRK channels expressed in Xenopus oocytes with or without their physiological activator, the G protein subunit dimer Gβγ. ETX binding site and mode of action were analysed using molecular dynamic (MD) simulations and kinetic modelling, and the predictions were tested by mutagenesis and functional testing. We show that ETX is a subunit-selective, allosteric blocker of GIRKs. The potency of ETX block is increased by Gβγ, in parallel with channel activation. MD simulations and mutagenesis locate the ETX binding site in GIRK2 to a region associated with phosphatidylinositol-4,5-bisphosphate (PIP2) regulation, and suggest that ETX acts by closing the helix bundle crossing (HBC) gate and altering channel's interaction with PIP2. The apparent affinity of ETX block is highly sensitive to changes in channel gating caused by mutations in Gβ1 or GIRK subunits. ETX block of GIRKs is allosteric, subunit-specific, and enhanced by Gβγ through an intricate network of allosteric interactions within the channel molecule. Our findings pose GIRK as a potential therapeutic target for ETX and ETX as a potent allosteric GIRK blocker and a tool for probing gating-related conformational changes in GIRK.

Exploring DL-2-aminobutyric acid: DFT analysis and spectroscopic characterization

DL-2-aminobutyric acid (α-aminobutyric acid), an alpha amino acid, has been extensively investigated through experimental and theoretical methods. Theoretical analyses have been carried out using density functional theory. Spectroscopic techniques such as Fourier transform infrared and UV spectroscopy have been employed, with experimental spectra aligning closely with theoretical predictions. Studies of electronic transitions between unoccupied and occupied states have been performed in solvents like dimethyl sulfoxide (DMSO) and Methanol (MeOH). The Time Dependent Density Functional Theory (TD-DFT) method and Iterative Electrostatic Potential Continuum Model (IEPCM) model assessed charge transfer in these solvents. Vibrational analysis and other studies, including Frontier Molecular Orbital (FMO), natural bond analysis, and non-linear optical analysis, were based on structures optimized using the B3LYP method with the 6-311++G(d,p) basis set. Total potential energy distribution analysis has been done for the detailed analysis of internal coordinates to each vibrational mode. The atom in molecule theory was applied via electron localization functions to examine ellipticity, binding energy, and isosurface projections. Natural bond analysis was conducted to investigate interference energy, bond hybridization, and interactions between donor and acceptor sites. Reactive sites were identified through Fukui functions and molecular electrostatic potential analysis. Thermodynamic properties, including free energy, enthalpy, entropy, internal energy, and heat capacities at various temperatures, were computed using the Atomistica online calculator. The electrophilicity index, derived from FMO analysis, suggested the compound’s potential bioactivity. Molecular docking studies with various proteins revealed the lowest binding energy of −4.9 kcal/mol. α-Aminobutyric acid and its derivatives exhibit similar Absorption, distribution, metabolism, and excretion (ADME) properties, indicating their potential utility in drug development. Minimal conformational changes in the peptide backbone after equilibrium have been explained by using molecular dynamics simulations, and molecular mechanics Poisson–Boltzmann surface area calculation showed an average free binding energy of −23.48 ± 1.72 kcal/mol.

Establishment of a Yeast Two-Hybrid-Based High-Throughput Screening Model for Selection of SARS-CoV-2 Spike-ACE2 Interaction Inhibitors

The recent coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has exerted considerable impact on global health. To prepare for rapidly mutating viruses and for the forthcoming pandemic, effective therapies targeting the critical stages of the viral life cycle need to be developed. Viruses are dependent on the interaction between the receptor-binding domain (RBD) of the viral Spike (S) protein (S-RBD) and the angiotensin-converting enzyme 2 (ACE2) receptor to efficiently establish infection and the following replicate. Targeting this interaction provides a promising strategy to inhibit the entry process of the virus, which in turn has both preventive and therapeutic effects. In this study, we developed a robust and straightforward assay based on the Yeast-Two Hybrid system (Y2H) for identifying inhibitors targeting the S-RBD-ACE2 interaction of SARS-CoV-2. Through high-throughput screening, two compounds were identified as potential entry inhibitors. Among them, IMB-1C was superior in terms of pseudovirus entry inhibition and toxicity. It could bind to both ACE2 and S-RBD and induce conformational change in the S-RBD+ACE2 complex. This is the first study to verify the feasibility of utilizing the Y2H system to discover potent SARS-CoV-2 inhibitors targeting the receptor recognition stage. This approach may also be applied in the discovery of other virus receptor recognition inhibitors.

Photophysical Properties, Fluorescence Quenching of Metformin Hydrochloride by Caffeine, and its Docking with the AMP-activated protein kinase receptor.

In this research, the photophysical properties of metformin hydrochloride (MF-HCl) were studied using spectroscopic and molecular docking techniques. The interaction between metformin hydrochloride and caffeine is essential for understanding the pharmacokinetics of metformin, particularly in populations with high caffeine consumption. Metformin is a first-line medication for managing type 2 diabetes, while caffeine is a widely consumed dietary stimulant. Knowing how caffeine may affect the action of metformin is crucial for effective diabetes management. The spectroscopic techniques results showed that the photophysical properties (fluorescence quantum yields, lifetime, radiative, and non-radiative decay) of the drug are influenced by solvent polarity and drug concentration. The binding mechanism of metformin hydrochloride-caffeine (MF-HCl-CAF) was identified through the fluorescence quenching method. The quenching of drugs induced by caffeine is due to ground state complex formation. The binding occurs due to hydrogen bonds and Van der Waals forces in the reaction. The förster resonance energy transfer (FRET) between metformin hydrochloride and caffeine was also calculated using flourtools.com software. The threshold distance (R0), for 50% energy transfer from metformin hydrochloride to caffeine is 1.81nm and the binding distance (r), between caffeine and the amino acid residue in metformin hydrochloride is 1.55nm. Dynamic light scattering (DLS), Zeta potential, and Fourier transform infrared (FTIR) spectroscopy confirm the conformational change of the drugs, as the caffeine molecule binds to metformin hydrochloride molecules. The molecular docking of metformin hydrochloride with the amp-activated protein kinase receptor (PDB Id: 1z0n) is analyzed. Again the docking of both metformin hydrochloride and caffeine (two ligands) with the protein receptor (PDB Id: 1z0n) was also analyzed and the results agreed with the fluorescence quenching techniques.

Excited-State Rotational Dynamics of Amine-Functionalized Terephthalic Acid Derivatives as Linker Models for Metal-Organic Frameworks.

Understanding how structural modifications affect the photophysics of organic linkers is crucial for their integration into metal-organic frameworks (MOFs) for light-driven applications. This study explores the impact of varying the amine functional group position on two terephthalic acid derivatives─linker 1 and linker 2─by investigating their photophysics through a combination of steady-state and ultrafast laser spectroscopy and time-dependent density functional theory (TD-DFT) calculations. With tetrahydrofuran as the solvent, time-correlated single-photon counting revealed a 2-fold increase in the S1 excited-state lifetime of the molecule with the amine group at the meta position compared with that of the molecule with the amine group at the ortho position. This phenomenon can be attributed to restricted intramolecular twisting for the molecule with the amine group in the meta position. In this regime, an interplay of high-energy steric and conjugation barriers was revealed for the molecule with the amine group at the meta position by TD-DFT calculations. Moreover, femtosecond/nanosecond transient absorption spectroscopy revealed a reversible excited-state conformational change for the ortho isomer via intramolecular rotation that occurred within ∼110 ps, unlocking a triplet state manifold. This study underscores the importance of modifying organic emitters, either as free linkers or within MOFs, to increase their performance in sensing and light-emitting applications.

Exploring the Gating Mechanism of the Human Copper Transporter, hCtr1, Using EPR Spectroscopy

Ctr1 is a membrane-spanning homotrimer that facilitates copper uptake in eukaryotic cells with high affinity. While structural details of the transmembrane domain of human Ctr1 have been elucidated using X-ray crystallography and cryo-EM, the transfer mechanisms of copper and the conformational changes that control the gating mechanism remain poorly understood. The role of the extracellular N-terminal domains is particularly unclear due to the absence of a high-resolution structure of the full-length hCtr1 protein and limited biochemical and biophysical characterization of the transporter in solution and in cell. In this study, we employed distance electron paramagnetic resonance to investigate the conformational changes of the extracellular N-terminal domain of full-length hCtr1, both in vitro and in cells, as a function of Cu(I) binding. Our results demonstrate that at specific Cu(I) concentrations, the extracellular chains move closer to the lumen to facilitate copper transfer. Additionally, while at these concentrations the intracellular part is penetrating the lumen, suggesting a ball-and-chain gating mechanism. Moreover, this phenomenon was observed for both reconstituted protein in micelles and in native cell membranes. However, the measured distance values were slightly different, suggesting that the membrane’s characteristics and therefore its lipid composition also impact and even regulate the gating mechanism of hCtr1.

Pretrained Deep Neural Network Kin-SiM for Single-Molecule FRET Trace Idealization.

Single-molecule fluorescence resonance energy transfer (smFRET) has emerged as a pivotal technique for probing biomolecular dynamics over time at nanometer scales. Quantitative analyses of smFRET time traces remain challenging due to confounding factors such as low signal-to-noise ratios, photophysical effects such as bleaching and blinking, and the complexity of modeling the underlying biomolecular states and kinetics. The dynamic distance information shaping the smFRET trace powerfully uncovers even transient conformational changes in single biomolecules both at or far from equilibrium, relying on trace idealization to identify specific interconverting states. Conventional trace idealization methods based on hidden Markov models (HMMs) require substantial a priori knowledge of the system under study, manual intervention, and assumptions about the number of states and transition probabilities. Here, we present a deep learning framework using long short-term memory (LSTM) to automate the trace idealization, termed Kin-SiM. Our approach employs neural networks pretrained on simulated data to learn high-order correlations in the multidimensional FRET trajectories. Without user input of Markovian assumptions, the trained LSTM networks directly idealize the FRET traces to extract the number of underlying biomolecular states, their interstate dynamics, and associated kinetic parameters. On benchmark smFRET data sets, Kin-SiM achieves a performance similar to conventional HMM-based methods but with less hands-on time and lower risk of bias. We further systematically evaluate the key training factors that affect network performance to define the correct hyperparameter tuning for applying deep neural networks to smFRET data analyses.

Eco-benign Synthesis of Multifunctional Silver Nanoparticles Using Leptochloa fusca for Enhanced Biomedical and Catalytic Applications.

Abstract The synthesis of nanomaterials with diverse biomedical and catalytic potentials is crucial in this era of rapidly advancing technology and increasing demand for innovative solutions in medicine and catalysis. The current study utilizes Leptochloa fusca aqueous extract for the eco-benign and cost-effective green synthesis of multifunctional silver nanoparticles (LF-AgNPs) with enormous potential of biomedical and catalytic applications. The bio-fabricated nanoparticles were characterized employing various analytical techniques including UV—Visible, FTIR, XRD, EDX, SEM and TGA. These analyses confirmed the synthesis, chemical composition, size, morphology, crystalline nature and thermal stability of LF-AgNPs. The surface plasmon resonance of LF-AgNPs was recorded at 432-436 nm and the particle size measured via SEM analysis was noted to be 40 to 75 nm. The synthesized LF-AgNPs were examined for their biomedical and catalytic applications. The binding affinity of LF-AgNPs with ssDNA and BSA was ratified spectroscopically by UV—Visible and FTIR techniques. The hyperchromism observed in the UV-Vis absorption spectra, as well as changes in the FTIR spectra of ssDNA and BSA upon interaction with LF-AgNPs, indicate the formation of LF-AgNPs—ssDNA and LF-AgNPs—BSA complexes. This type of binding results in conformational changes in ssDNA and BSA structures and therefore LF-AgNPs hold potential for numerous applications like drug delivery, anticancer agents and receptor targeting. The biosynthesized LF-AgNPs were also found to have effective antioxidant (70%) and H2O2 scavenging (77%) properties. Additionally, the agar well diffusion technique demonstrated efficient antibacterial efficacy for LF-AgNPs against both Gram +ve (Staphylococcus aureus) and Gram -ve (Klebsiella pneumoniae) strains with ZOI values of 19 ± 0.7 mm and 19 ± 0.5 mm, respectively. Moreover, LF-AgNPs efficiently catalysed the reduction of methylene blue in less than 9 minutes, using NaBH4 as a reducing agent. Therefore, LF-AgNPs could find potential application in catalytic and biomedical fields.&amp;#xD;

Phosphorylation of Bok at Ser-8 blocks its ability to suppress IP3R-mediated calcium mobilization

BackgroundBok is a poorly characterized Bcl-2 protein family member with roles yet to be clearly defined. It is clear, however, that Bok binds strongly to inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), which govern the mobilization of Ca2+ from the endoplasmic reticulum, a signaling pathway required for many cellular processes. Also known is that Bok has a highly conserved phosphorylation site for cAMP-dependent protein kinase at serine-8 (Ser-8). Whether Bok, or phosphorylated Bok, has any direct impact on the Ca2+ mobilizing function of IP3Rs remains to be established.MethodsBok Ser-8 phosphorylation was characterized using purified proteins, G-protein coupled receptor agonists that increase cAMP levels in intact cells, mass spectrometry, and immunoreactivity changes. Also, using mammalian cells that exclusively or predominately express IP3R1, to which Bok binds strongly, and a fluorescent Ca2+-sensitive dye or a genetically-encoded Ca2+ sensor, we explored how endogenous and exogenous Bok controls the Ca2+ mobilizing function of IP3R1, and whether Bok phosphorylation at Ser-8, or replacement of Ser-8 with a phosphomimetic amino acid, is regulatory.ResultsOur results confirm that Ser-8 of Bok is phosphorylated by cAMP-dependent protein kinase, and remarkably that phosphorylation can be detected with Bok specific antibodies. Also, we find that Bok has suppressive effects on IP3R-mediated Ca2+ mobilization in a variety of cell types. Specifically, Bok accelerated the post-maximal decline in G-protein coupled receptor-induced cytosolic Ca2+ concentration, via a mechanism that involves suppression of IP3R-dependent Ca2+ release from the endoplasmic reticulum. These effects were dependent on the Bok-IP3R interaction, as they are only seen with IP3Rs that can bind Bok (e.g., IP3R1). Surprisingly, Bok phosphorylation at Ser-8 weakened the interaction between Bok and IP3R1 and reversed the ability of Bok to suppress IP3R1-mediated Ca2+ mobilization.ConclusionsFor the first time, Bok was shown to directly suppress IP3R1 activity, which was reversed by Ser-8 phosphorylation. We hypothesize that this suppression of IP3R1 activity is due to Bok regulation of the conformational changes in IP3R1 that mediate channel opening. This study provides new insights on the role of Bok, its interaction with IP3Rs, and the impact it has on IP3R-mediated Ca2+ mobilization.

Conformational Engineering of Flexible Protein Fragments on the Surface of Different Nanoparticles: The Surface-Atom Mobility Rules.

As a newly emerging technology, conformational engineering (CE) has been gradually displaying the power of producing protein-like nanoparticles (NPs) by tuning flexible protein fragments into their original native conformation on NPs. But apparently, not all types of NPs can serve as scaffolds for CE. To expedite the CE technology on a broader variety of NPs, the essential characteristic of NPs as scaffolds for CE needs to be identified. Herein, we investigate the potential of two distinct types of NPs as scaffolds for CE: CdSe/ZnS quantum dots (QDs), an ionic compound NP, and palladium NPs (PdNPs), a metal NP. The results demonstrate that while QDs cannot support the restoration of the native conformation and function of the complementary-determining region (CDR) fragments of antibodies, PdNPs can. The notably disparate outcomes unequivocally show that the mobility of the surface atoms/adatoms of the NPs or the mobility of the conjugating bonds to the NPs is essential for CE, which allows the conjugated peptides to undergo a conformational change from their initial random conformation to their most stable native conformation under the constraints mimicking the native long-range interactions in the original proteins. This discovery opens the door for CE on more NPs in the future.

In-silico study of E169G and F242K double mutations in leucine-rich repeats (LRR) polygalacturonase inhibiting protein (PGIP) of Gossypium barbadense and associated defense mechanism against plant pathogens

BackgroundPolygalacturonase inhibiting proteins (PGIPs) play a pivotal role in plant defense against plant pathogens by inhibiting polygalacturonase (PG), an enzyme produced by pathogens to degrade plant cell wall pectin. PGIPs, also known as leucine-rich repeat pathogenesis-related (PR) proteins, activate the host’s defense response upon interaction with PG, thereby reinforcing the host defense against plant pathogens attacks. In Egyptian or extra-long staple cotton (Gossypium barbadense), the interaction between PGIP and PG is one of the crucial steps in the defense mechanism against major pathogens such as Xanthomonas citri pv. malvacearum and Alternaria macrospora, which are responsible for bacterial leaf blight and leaf spot diseases, respectively.ResultsTo unravel the molecular mechanisms underlying these PR proteins, we conducted a comprehensive study involving molecular modeling, protein-protein docking, site-specific double mutation (E169G and F242K), and molecular dynamics simulations. Both wild-type and mutated cotton PGIPs were examined in the interaction with the PG enzyme of a bacterial and fungal pathogen. Our findings revealed that changes in conformations of double-mutated residues in the active site of PGIP lead to the inhibition of PG binding. The molecular dynamics simulation studies provide insights into the dynamic behaviour and stability of the PGIP-PG complexes, shedding light on the intricate details of the inhibitory and exhibitory mechanism against the major fungal and bacterial pathogens of G. barbadense, respectively.ConclusionsThe findings of this study not only enhance our understanding of the molecular interactions between PGs of Xanthomonas citri pv. malvacearum and Alternaria macrospora and PGIP of G. barbadense but also present a potential strategy for developing the disease-resistant cotton varieties. By variations in the binding affinities of PGs through specific mutations in PGIP, this research offers promising avenues for the development of enhanced resistance to cotton plants against bacterial leaf blight and leaf spot diseases.