Search Results (203)

Intrinsically disordered regions enable transcription factors (TFs) to undergo structural changes upon ligand binding, facilitating the transduction of environmental signals into gene expression. In this study, we applied molecular modeling methods to explore the hypothesis that unstructured inter-domain and subdomain linkers in bacterial TFs can function as sensors for carbohydrate signaling molecules. We combined molecular dynamics simulations and carbohydrate docking to analyze six repressors with GntR-type DNA-binding domains, including UxuR, GntR and FarR from Escherichia coli, as well as AraR, NagR and YydK from Bacillus subtilis. Protein models obtained from different time points of the dynamic simulations were subjected to sequential carbohydrate docking. We found that the inter-domain linker of the UxuR monomer binds D-fructuronate, D-galacturonate, D-glucose, and D-glucuronate with an affinity comparable to nonspecific interactions. However, these ligands formed multimolecular clusters, a feature absent in the UxuR dimer, suggesting that protein dimerization may depend on linker occupancy by cellular carbohydrates. D-glucose interacted with linkers connecting subdomains of the LacI/GalR-type E-domains in GntR and AraR, forming hydrogen bonds that connected distant structural modules of the proteins, while in NagR, FarR and YydK, it bridged the inter-domain linkers and a β-sheet within the HutC-type E-domains. Hence, our results establish flexible linkers as pivotal metabolic sensors that directly integrate nutritional cues to alter gene expression in bacteria. Full article

Vascular endothelial growth factor 165 (VEGF₁₆₅) stands out as a pivotal isoform of the VEGF-A protein and is critically involved in various angiogenesis-related diseases. Consequently, it has emerged as a promising target for diagnosing and treating such conditions. Structurally, VEGF₁₆₅ forms a homodimer, and each of its constituting monomers comprises a receptor-binding domain (RBD) and a heparin-binding domain (HBD). These two domains are linked by a flexible linker, and thus the overall structure of VEGF₁₆₅ remains incompletely understood. Aptamers are known as potent drugs that interact with VEGF₁₆₅, and dimeric aptamers that can simultaneously interact with two distant domains are frequently adopted to improve the potency. However, designing such aptamer dimers faces challenges in regard to determining the appropriate length of the linker connecting the two aptamer fragments. To gain insight into this distance information, we here employ biased molecular dynamics (MD) simulations with the umbrella sampling method, with the distance between the two HBDs serving as a reaction coordinate. Our simulations reveal an overall preference for compact conformations with HBD-HBD distances below 3 nm, with the minimum of the potential of mean force located at 1.1 nm. We find that VEGF₁₆₅ with the optimal HBD-HBD distance forms hydrogen bonds with its receptor VEGFR-2 that well match experimentally known key hydrogen bonds. We then try to computationally design aptamer homodimers consisting of two del5-1 aptamers connected by various linker lengths to target VEGF₁₆₅. Collectively, our findings may provide quantitative guidelines for rationally designing high-affinity aptamers for targeting VEGF₁₆₅. Full article

A conjugated helical cage, comprising two 1,3,5-tris(phenylethynyl)benzene units connected by diyne linkers, was successfully synthesized. X-ray crystallography revealed helical molecular structures with large twisted angles and a 1:1 mixture of P- and M-enantiomers. Variable-temperature-NMR measurement indicated the racemization process between the enantiomers occurs rapidly on the NMR timescale. The rapid interconversion is attributed to the flexible diyne linkages, even though they were believed to be rigid. Full article

Four new porous homochiral metal–organic frameworks (MOFs), [M₂(camph)₂(bpa)]∙Solv (M = Co(II), Ni(II), Cu(II) and Zn(II)), based on (+)-camphoric acid (H₂camph) and 1,2-bis(4-pyridyl)ethane (bpa) were synthesized and characterized. The crystal structures of [Ni₂(camph)₂(bpa)] and [Zn₂(camph)₂(bpa)] were established by single-crystal X-ray diffraction analysis. Powder X-ray data prove the phase purity and isostructural nature of all four compounds. The thermal stability of [M₂(camph)₂(bpa)] was found to depend on the electronic configuration, as well as on the redox properties of the metal cation, and varied from 225 °C (M = Zn²⁺) to 375 °C (M = Ni²⁺). The reversible, solvent-induced sponge-like dynamics of the coordination frameworks was thoroughly investigated. Changes in the positions of reflexes, related to the length of the flexible bpa linker, were observed by powder XRD, pointing to transitions between an open-framework phase and a squeezed, non-porous phase in a crystal-to-crystal manner, while the integrity and connectivity of the coordination network were maintained. Size-selective adsorption from a benzene–cyclohexane 1:1 mixture on [Zn₂(camph)₂(bpa)] was studied by ¹H NMR analysis. The benzene-favorable composition of guest molecules (C₆H₆:C₆H₁₂ = 5:1) occluded within the host crystalline sponge revealed a preferable adsorption affinity towards smaller benzene compared with larger cyclohexane. High framework stability in various solvents, as well as successful molecular separation in the liquid state, validates the potential utilization of chiral porous metal(II) camphorate MOFs in important stereoselective applications. Full article

Tricopeptide repeats refer to 30 or more amino acid (aa) repeats, of which the best studied ones are 34-aa and 35-aa long, named Tetratricopeptide and Pentatricopeptide repeats, respectively, and abbreviated as TPR and PPR. Recently, 37-aa and 38-aa repeats (Heptatricopeptide, HPR; Octatricopeptide, OPR) have been reported, but 36-aa repeats or repeats outside the 34–38 range (such as 33-aa or 39-aa) are apparently missing. This review is an analytical discourse of the structural and functional commonalities as well as differences among all tricopeptide repeats. In structure, the use of Artificial Intelligence (AI)-based prediction and experimental 3D structures revealed that regardless of the number of amino acids, these repeats are all alpha-helical in nature, whereby the tandem helices are joined by relatively flexible linkers or spacers to form a superhelix. In function, many tricopeptide repeats bind specific RNA, thus playing important roles in RNA processing and stability. The specificity is determined by the interaction between specific amino acid residues with the nucleotides in the RNA, while the helices offer a scaffold that holds the interacting residues in position. Detailed analysis of various known TPR and PPR revealed conserved amino acids at specific positions, such that they serve as signature motifs. Moreover, extra helices upstream or downstream of the repeat domains often maintain the continuum of the superhelical vortex. Evidently, the overall helicity and the presence of critical amino acid residues in strategic places are more important for the biological function of the tricopeptide repeats than the exact amino acid length of the repeat. Full article

Polymyxin antibiotics are often the last line of defense against multidrug-resistant Gram-negative pathogens. A key resistance mechanism involves the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to lipid A, mediated by the bifunctional enzyme ArnA. However, the evolutionary rationale and structural basis for ArnA’s domain fusion, hexameric assembly, and catalytic coordination remain mechanistically unresolved. Here, we integrate evolutionary genomics, high-resolution cryo-electron microscopy (cryo-EM), and computational protein design to provide a comprehensive mechanistic analysis of ArnA. Our evolutionary analysis reveals that the dehydrogenase (DH) and formyltransferase (TF) domains evolved independently and were selectively fused in Gammaproteobacteria, suggesting an adaptive advantage. A 2.89 Å cryo-EM structure of apo-ArnA resolves the flexible interdomain linker and reveals a DH-driven hexameric architecture essential for enzymatic activity. 3D variability analysis captures intrinsic conformational dynamics, indicating a molecular switch that may coordinate sequential catalysis and substrate channeling. Structure-based peptide inhibitors targeting the hexamerization and predicted ArnA–ArnB interaction interfaces were computationally designed, offering a novel strategy for disrupting L-Ara4N biosynthesis. These findings illuminate a previously uncharacterized structural mechanism of antimicrobial resistance and lay the groundwork for therapeutic intervention. Full article

Single-molecule nanopore sensors have enabled real-time detection of enzymatic cleavage events, yet achieving sensitive and specific analysis of protease activity remains an important challenge for diagnostic applications. We engineered OmpG nanopore constructs incorporating thrombin recognition peptides into loop 6 with varied flexible and negatively charged linkers to optimize accessibility and cleavage. SDS-PAGE gel analysis showed that constructs with the recognition peptide placed after residue D225 and incorporating dual linkers achieved cleavage efficiencies up to 95%, whereas constructs without linkers showed limited cleavage. Single-channel recordings revealed that linker integration modulates pore conductance, with extended loops exhibiting intermediate open-state currents near 18 pA compared to 25 pA in wild-type OmpG. Upon thrombin addition, rapid and irreversible current drops confirmed real-time protease activity detection. These results demonstrate the critical role of linker design, particularly flexibility and charge, in optimizing nanopore protease sensors, providing a versatile platform for biomedical applications. Full article

Currently, a major challenge exists in CRISPR-mediated genome editing research in sheep: the low efficiency of exogenous large DNA fragment targeted integration without drug selection or fluorescence enrichment. This restriction significantly impedes the use of precise genome editing in sheep for agricultural, biological, and biomedical purposes. In this study, we employed the strategy of increasing the local concentration of the homologous repair template at the site of the DNA double-strand break (DSB). We achieved this by fusing the DNA binding domain (BD) of the Gal4 protein (Gal4-BD) to the N-terminal end of the SpCas9 protein using a 32-amino acid (aa) flexible linker. Additionally, we incorporated a 17 bp UAS at the 3′ end of the donor template, which can be specifically recognized and bound by Gal-BD. As a result, we observed a significant improvement in the knock-in efficiency of the exogenous large DNA fragment (2997 bp) in sheep fetal fibroblasts (SFFs), increasing it from 5.30% (8/151) to 16.67% (32/192), providing a comparatively efficient and user-friendly method to promote CRISPR-mediated gene knock-in in sheep. Full article

Background: Enterotoxigenic Escherichia coli (ETEC) is a zoonotic pathogen causing diarrhea and mortality in infants and livestock. Its numerous serotypes necessitate the urgent development of multivalent vaccines for effective prevention, thereby reducing public health and economic threats. Methods: Computational bioinformatics analyses were conducted on five major ETEC adhesins structural subunits (FaeG, FanC, FasA, FimF41a, and FedF). Dominant epitopes were selected and concatenated via flexible linkers, incorporating the PADRE sequence and LTb adjuvant to design a multi-epitope fusion antigen (MEFA). The recombinant MEFA protein was expressed in a prokaryotic system. Furthermore, molecular dynamics simulations, docking, and immune simulations assessed structural stability and immunogenicity. Immunoreactivity was tested by Western blot. Murine immunization evaluated antibody responses, lymphocyte proliferation, cytokine secretion, and protection against ETEC challenge. Results: Structural modeling showed an extended conformation, with docking and simulations indicating strong immune activation. Western blot confirmed MEFA immunoreactivity. MEFA induced high antigen-specific antibody titers, enhanced splenocyte proliferation, and increased IFN-γ and IL-4 secretion, indicating a Th2-biased response in mice. Vaccinated mice survived lethal ETEC challenge and maintained intestinal integrity. Conclusions: The MEFA candidate vaccine effectively induces robust humoral and cellular immune responses and provides protection against ETEC infection, representing a promising strategy for next-generation multivalent ETEC vaccines. Full article

Acquired Immunodeficiency Syndrome (AIDS) is caused by Human Immunodeficiency Virus (HIV), and continues to be responsible for a substantial number of deaths worldwide each year. Development of a robust and efficient HIV-1 vaccine remains a critical priority. Structural analysis of viral proteins provides a foundational approach to designing peptide-based immunogenic vaccines. In the current experiment, we used computational prediction approaches alongside molecular docking and molecular dynamics (MD) simulations to identify potential epitopes within gp120 and Nef proteins. The selected co-epitopes were fused with the HBsAg-binding protein (SBP), a 344-amino acid protein previously identified in our laboratory through screening of a human liver cDNA expression library against HBsAg, to facilitate efficient delivery to and uptake by dendritic cells (DCs), thereby enhancing antigen (Ag) presentation. Flexible linkers are used to connect B cells, Helper T Lymphocytes (HTLs), and Cytotoxic T Lymphocytes (CTLs) in a sequential manner. The assembled vaccine construct comprises 757 amino acids, corresponding to a recombinant protein of 83.64 kDa molecular weight. Structural analysis through docking studies, MD simulations, and 3D structure validation revealed that the designed protein exhibits high structural stability and potential for interaction with Toll-like receptors (TLRs). These findings support the vaccine’s ability to enhance cellular and humoral feedback, including the stimulation of T and B cells and induction of antibody (Ab) production. The results underscore the promise of this in silico designed co-epitope vaccine as a viable candidate for HIV-1 prevention and suggest that such constructs may serve as effective immunogens in future HIV-1 vaccine strategies. Full article

BACH1 (BTB And CNC Homology 1) and NRF2 (Nuclear Factor Erythroid 2-related Factor 2) are transcription factors that regulate antioxidant and iron metabolism genes by competing for binding to cis-regulatory Maf-binding elements (CsMBEs) as heterodimers with small Maf proteins (sMafs). To dissect the mechanisms underlying this competition, we developed a chimeric tethering system where the DNA-binding domains of BACH1 or NRF2 were covalently linked to sMafG via a flexible, cleavable linker. This design enables efficient heterodimer formation on DNA and circumvents kinetic barriers to partner exchange in the solution. The site-specific fluorescent labelling of proteins allowed for the tracking of complex compositions by electrophoretic mobility shift assays. Both BACH1/sMafG and NRF2/sMafG heterodimers bind CsMBEs with similar affinities. Notably, DNA binding by BACH1 was impaired in a C574-dependent, redox-sensitive manner and promoted the exchange of heterodimer partners. Competition assays demonstrated that BACH1 and NRF2 can displace each other from preformed DNA-bound complexes, with greater efficiency when presented as preassembled heterodimers with sMafG. These findings reveal a redox-sensitive mechanism for regulating transcriptional switches at CsMBEs and highlight how preformed heterodimers facilitate the rapid displacement at target promoters. Full article

Near-ultraviolet photoluminescence liquid-crystalline molecules (PLLCs) have attracted attention for temperature-responsive photoluminescence (PL) modulation and ON/OFF sensing under external stimuli. We recently developed mesogenic dimers composed of two hexyloxy-substituted, fluorinated tolane-type cores linked by alkylene-1,n-dioxy chains that exhibited near-UV PL in the solid state. However, the formation of LC phases and the temperature range of the LC state were limited. To improve LC phase stability, in this study, we extended the flexible terminal chains and repositioned the fluorinated aromatic rings from the outer to the inner core positions. Accordingly, we synthesized mesogenic dimers with even-numbered alkylene-1,n-dioxy linkers (hexylene, octylene, and decylene) and outer- or inner-ring fluorination. Outer-ring fluorination led to high melting temperatures and stable crystalline phases with limited mesophase formation. In contrast, inner-ring fluorination induced nematic phases upon heating and cooling owing to zig-zag molecular structures that disrupted crystallinity. Photophysical studies confirmed near-UV PL in solution and solid states; however, the quantum yield of the solution PL was low (<0.01). In the solid state, the PL efficiencies and wavelengths were influenced by the fluorinated aromatic ring position and linker length. This study provides important molecular design criteria for developing stable LC materials with tunable near-UV luminescence for temperature-responsive optical devices. Full article

PROTACs are trimeric small molecules consisting of a specific modulator of the target protein connected to a ligase-recruiting ligand via a suitably flexible linker. Ligase-recruiting ligands deliver ubiquitin ligases like E3 ligase to the Protein of Interest (POI). The vicinity of the POI-PROTAC-E3 ternary complex enables the E3 ligase to ubiquitinate the surface lysine residues of the POI. The Ubiquitin–Proteasome System (UPS) then degrades the POI. However, despite the considerable advances in the design of PROTACs targeting several types of enzymes and receptors, this strategy is still facing the challenges of precision target delivery and duration of action. In this review, we highlight the recent approaches for the development of PROTAC prodrugs or pro-PROTAC to control the delivery of PROTACs and achieve the required on-target exposure. This strategy may facilitate the application of the PROTAC technology and expand its clinical benefits. Full article

A novel series of multifunctional tacrine–quinoline hybrids were designed, synthesized, and evaluated as potential anti-Alzheimer’s agents. These compounds incorporate tacrine for cholinesterase’s inhibition and 8-hydroxyquinoline for metal chelation. Piperazine was selected as a linker to provide conformational flexibility and to create favorable cation–π interactions with residues in the mid-gorge region of AChE, enhancing dual-site binding with AChE to inhibit Aβ aggregation. In vitro studies demonstrated submicromolar inhibitory activity toward both AChE and BuChE, particularly for compounds 16e (IC₅₀ = 0.10 μM for AChE, 0.043 μM for BuChE) and 16h (IC₅₀ = 0.21 μM for AChE, 0.10 μM for BuChE). These compounds also exhibited potent inhibition of self-induced Aβ_1–42 aggregation (16e: 80.5% ± 4.4%, 16h: 93.2% ± 3.9% at 20 μM). Kinetic analyses revealed mixed-type inhibition, suggesting dual binding to both CAS and PAS of AChE. UV–vis spectrometry confirmed the chelation of Cu²⁺ and Zn²⁺ ions by the 8-hydroxyquinoline moiety. These findings highlight the tacrine–quinoline scaffold as a promising platform for the discovery of a multitarget-directed anti-AD drug. Full article

Through-space charge transfer (TSCT) between spatially adjacent donor and acceptor units has garnered considerable attention as a promising design principle for optoelectronic materials. While TSCT systems incorporating rigid spacers have been extensively studied to enhance through-space interactions, transition metal complexes connected by flexible linkers remain underexplored, despite increasing interest in their potential TSCT behavior. Herein, we report the design and synthesis of a donor–acceptor–donor (D-A-D)-type complex (1), in which a central naphthalenediimide (NDI) electron acceptor is linked to 2-phenylpyridinato(salicylaldiminato)platinum(II) complexes via flexible alkyl linkers. By systematically varying the linker length (n = 3, 4, 5, 6; 1a–d), we achieved precise control over the spatial arrangement between the NDI core and the platinum moieties in solution. Notably, compound 1a (n = 3) adopts an S-shaped conformation in solution, giving rise to a distinct TSCT absorption band. The structural and photophysical properties were thoroughly investigated using single-crystal X-ray diffraction, ¹H NMR, NOESY analysis, and DFT calculations, which collectively support the existence of the folded conformation and associated TSCT behavior. These findings highlight that TSCT can be effectively induced in flexible molecular systems by exploiting intramolecular spatial proximity and non-covalent interactions, thereby offering new avenues for the design of responsive optoelectronic materials. Full article