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Pinpointing the l-phenylalanine binding sites of TyrR using biosensors and computer-aided simulation
摘要： Objectives To determine the binding sites for L-phenylalanine in TyrR protein via a rational mutation analysis combining biosensors and computer-aided simulation. Results TyrR protein of Escherichia coli is the chief transcriptional regulator of several genes essential for the biosynthesis and transport of aromatic amino acids. The identification of ligand-binding sites is often the starting point for protein function annotation and structure-based protein design. Here we combined computer-aided prediction methods and biosensors to identify the ligand-binding sites for L-Phe in TyrR protein. Conclusions Residues at positions 160, 173 and 184 of TyrR protein are important for transcriptional activation of target genes tyrP induced by L-Phe, which indicates that they are the bona fide L-Phe binding sites of TyrR protein.
关键词： L-Phe binding site,Computer-aided simulation,TyrR protein,Biosensor
更新于2025-09-19 17:15:36
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Maintenance of Rhodopsin levels in <i>Drosophila</i> photoreceptor and phototransduction requires Protein Kinase D
摘要： During Drosophila phototransduction, the G protein coupled receptor (GPCR) Rhodopsin (Rh1) transduces photon absorption into electrical signal via G-protein coupled activation of phospholipase C (PLC). Rh1 levels in the plasma membrane are critical for normal sensitivity to light. In this study, we report that Protein kinase D (dPKD) regulates Rh1 homeostasis in adult photoreceptors. Although eye development and retinal structure are unaffected in the dPKD hypomorph (dPKDH), it exhibited elevated levels of Rh1. Surprisingly, despite having elevated levels of Rh1, no defect was observed in the electrical response to light in these flies. By contrast the levels of another transmembrane protein of the photoreceptor plasma membrane, Transient receptor potential (TRP) was not altered in dPKDH. Our results indicate that dPKD is dispensable for eye development but is required for maintaining Rh1 levels in adult photoreceptors.
关键词： Electroretinogram (ERG),Phototransduction,Retinal degeneration,Rhodopsin,Drosophila,Rhodopsin loaded vesicle (RLVs),Protein Kinase D
更新于2025-09-19 17:15:36
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Photocaged Quinone Methide Cross-linkers for Light-controlled Chemical Cross-linking of Protein-protein and Protein-DNA Complexes
摘要： Small molecule cross-linkers are invaluable for probing biomolecular interactions and for cross-linking mass spectrometry (CXMS) in addressing large protein complexes and intrinsically disordered proteins. Existing chemical cross-linkers target only a small selection of amino acid residues, limiting the number and type of cross-links, while conventional photocross-linkers target virtually all residues non-selectively, complicating data analysis. Here we report photocaged quinone methide (PQM)-based cross-linkers that are able to multitarget nine nucleophilic residues through specific Michael addition. In addition to Asp, Glu, Lys, Ser, Thr, and Tyr, PQM cross-linkers notably cross-linked Gln, Arg, and Asn hitherto untargetable by existing chemical cross-linkers, markedly increasing the number of residues targetable with a single cross-linker. Such multiplicity of cross-links will increase the abundance of cross-linked peptides for CXMS identification and afford ample constraints to facilitate structural deciphering. PQM cross-linkers were used in vitro, in E. coli, and in mammalian cells to cross-link dimeric proteins and endogenous membrane receptors. The cross-linker NHQM could directly cross-link proteins to DNA, for which few cross-linkers exist. The photoactivatable and multitargeting reactivity of these PQM cross-linkers will substantially enhance chemical cross-linking based technologies for studies of protein-protein and protein-DNA networks and for structural biology.
关键词： DNA-protein interaction,cross-linker,protein-protein interaction,photo-cross-linking,quinone methide
更新于2025-09-19 17:13:59
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The Kinetic and Molecular Basis for the Interaction of LexA and Activated RecA Revealed by a Fluorescent Amino Acid Probe
摘要： The bacterial DNA damage response (the SOS response) is a key pathway involved in antibiotic evasion and a promising target for combating acquired antibiotic resistance. Activation of the SOS response is controlled by two proteins: the repressor LexA and the DNA damage sensor RecA. Following DNA damage, direct interaction between RecA and LexA leads to de-repression of the SOS response. However, the exact molecular details of this interaction remain unknown. Here, we employ the fluorescent unnatural amino acid acridonylalanine (Acd) as a minimally-perturbing probe of the E. coli RecA:LexA complex. Using LexA labeled with Acd, we report the first kinetic model for the reversible binding of LexA to activated RecA. We also characterize the effects that specific amino acid truncations or substitutions in LexA have on RecA:LexA binding strength, and demonstrate that a mobile loop encoding LexA residues 75?84 comprises a key recognition interface for RecA. Beyond insights into SOS activation, our approach also further establishes Acd as a sensitive fluorescent probe for investigating the dynamics of protein-protein interactions in other complex systems.
关键词： SOS response,fluorescent amino acid probe,RecA,LexA,protein-protein interactions
更新于2025-09-19 17:13:59
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Analysis on FM-to-AM conversion of SSD beam induced by etalon effect in a high-power laser system
摘要： Malondialdehyde (MDA) was selected to represent a secondary by-product of lipid peroxidation during rice ageing. This study aimed to investigate the effects of MDA modification on the structural characteristics of rice protein. The results showed that as MDA concentration increased, rice protein carbonyl and disulphide groups increased, but sulphydryl content decreased. The blue shift of maximum fluorescence peak, the decrease of rice protein intrinsic fluorescence intensity and the reduction of surface hydrophobicity indicated the formation of protein aggregates caused by MDA oxidative modification. The results of molecular weight distribution and particle size distribution showed that MDA modification resulted in the formation of soluble protein aggregates, and the decrease of rice protein solubility indicated that insoluble protein aggregates were formed. Results of protein electrophoresis showed that MDA modification contributed to rice protein aggregation via non-disulphide covalent bonds. The results showed that rice protein gradually aggregated with increasing MDA concentration.
关键词： Aggregation,rice protein,protein oxidation,lipid peroxidation,rice ageing,malondialdehyde
更新于2025-09-19 17:13:59
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Azo-PROTAC: novel light-controlled small-molecule tool for protein knockdown
摘要： Reversibly altering endogenous protein levels are persistent issues. Herein, we designed photoswitchable Azo-PROTACs by including azobenzene moieties between ligands for the E3 ligase and the protein of interest. Azo-PROTACs are light-controlled small-molecule tools for protein knockdown in cells. The light-induced configuration change can switch the active state to induce protein degradation activity, which can be reversely controlled by light exposure in intact cells. We compared the protein degradation abilities of Azo-PROTACs with different configurations and linker lengths. Using the stable form with the best degradation ability against the BCR-ABL fusion and ABL proteins in myelogenous leukemia K562 cells, we showed that Azo-PROTAC combines the potent protein knockdown and facile cell uptake properties of the small-molecule PROTAC with a reversible photo-switch ability, offering a promising chemical knockdown strategy based on the light-induced reversible on/off property.
关键词： protein modifications,photoswitchable PROTAC,protein knockdown,ubiquitin-proteasome system.
更新于2025-09-19 17:13:59
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In Situ Investigation on the Protein Corona Formation of Quantum Dots by Using Fluorescence Resonance Energy Transfer
摘要： A fundamental understanding of nanoparticle–protein corona and its interactions with biological systems is essential for future application of engineered nanomaterials. In this work, fluorescence resonance energy transfer (FRET) is employed for studying the protein adsorption behavior of nanoparticles. The adsorption of human serum albumin (HSA) onto the surface of InP@ZnS quantum dots (QDs) with different chirality (d- and l-penicillamine) shows strong discernible differences in the binding behaviors including affinity and adsorption orientation that are obtained upon quantitative analysis of FRET data. Circular dichroism spectroscopy further confirms the differences in the conformational changes of HSA upon interaction with d- and l-chiral QD surfaces. Consequently, the formed protein corona on chiral surfaces may affect their following biological interactions, such as possible protein exchange with serum proteins plasma as well as cellular interactions. These results vividly illustrate the potential of the FRET method as a simple yet versatile platform for quantitatively investigating biological interactions of nanoparticles.
关键词： serum proteins,quantum dots,chirality,nanoparticle–protein interactions,protein corona,fluorescence resonance energy transfer
更新于2025-09-19 17:13:59
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Gambogic Acid Augments Black Phosphorus Quantum Dots (BPQDs)-Based Synergistic Chemo-Photothermal Therapy through Downregulating Heat Shock Protein Expression
摘要： In an attempt to attain synergistic therapeutic benefits and address various intrinsic limitations of the highly efficient black phosphorus quantum dots (BPQDs), we fabricated poly(L-lactide)-poly(ethylene glycol)-poly(L-lactide) triblock copolymer (PLLA-PEG-PLLA)-based nanocomposites co-loaded with BPQDs and gambogic acid (GA) using the supercritical carbon dioxide (SC-CO2) technology to achieve photoacoustic (PA) imaging-guided synergistic chemo-photothermal therapy. On the one hand, BPQDs displayed near-infrared (NIR)-induced hyperthermia through the high photothermal conversion efficiency. On the other hand, the NIR-responsive release of GA facilitated early apoptosis through specific binding to stress-induced overexpression of heat shock protein (HSP)-90 for combating thermoresistant tumor cells. GA significantly promoted the photothermal therapy (PTT) efficiency by enhancing both early and late apoptosis of BPQDs. Moreover, the encapsulation of BPQDs in the polymer significantly improved their chemical as well as photothermal stabilities. Our findings suggested that these nanocomposites fabricated using the eco-friendly supercritical fluid (SCF) technology provided good protection to the biodegradable BPQDs, offering a great potential towards cancer ablation through augmented synergistic effects.
关键词： Synergism,NIR-responsive,Black phosphorus,Heat shock protein,Photothermal therapy
更新于2025-09-19 17:13:59
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Study on Irradiation Effect of Mid-Infrared Free Electron Laser on Hen Egg-White Lysozyme by Using Terahertz-Time Domain Spectroscopy and Synchrotron-Radiation Vacuum-Ultraviolet Circular-Dichroism Spectroscopy
摘要： Mid-infrared free-electron laser (MIR-FEL) is potentially applicable for tissue ablation and dissociation of pathological peptide aggregates in medicine. However, it is still poorly understood how the MIR-FEL irradiation influences on functional proteins such as enzymes. In the current study, the effect of MIR-FEL on both aggregate and non-aggregate (= native) states of hen egg-white lysozyme (HEWL) as a representative enzyme has been investigated. Absorption intensity at terahertz region (0.3–1.2 THz, 10–40 cm?1) of the aggregate of HEWL was lower than that of the native HEWL, but the former was increased nearly to the same level with the latter after the MIR-FEL irradiation tuned to 6 μm that corresponds to carbonyl stretching vibrational mode of amide bonds (amide I). This indicates that the aggregate of HEWL was converted to the native state by the irradiation. On the other hand, synchrotron-radiation vacuum-ultraviolet circular-dichroism spectroscopy showed that protein conformation of the native HEWL, which was rich in α-helix, was little changed after the MIR-FEL irradiation under the same condition with the case of the aggregate of HEWL. Furthermore, the enzymatic hydrolysis activity of the native HEWL against bacterial glycan was not remarkably decreased by the irradiation. Therefore, it can be estimated that the native structure of HEWL is little damaged although the aggregate state can be easily dissociated by the resonant excitation at amide bonds.
关键词： Vacuum-ultraviolet circular-dichroism spectroscopy,Mid-infrared free-electron laser,Protein aggregate,Terahertz-time domain spectroscopy
更新于2025-09-19 17:13:59
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Detection and Quantification of HspX Antigen in Sputum Samples Using Plasmonic Biosensing: Toward a Real Point-of-Care (POC) for Tuberculosis Diagnosis
摘要： Advances occurred during the last years in the diagnosis of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis infection, have prompted increased survival rates of patients. However, limitations related to the inefficiency of an early detection still remain: some techniques and laboratory methods do not have enough specificity, most instruments are expensive, and require handling by trained staff. In order to contribute to a prompt and effective diagnosis of tuberculosis, we report the development of a portable, user-friendly and low-cost biosensor device for its early detection. By using a label-free Surface Plasmon Resonance (SPR) biosensor, we have established a direct immunoassay for the direct detection and quantification of the Heat shock protein X (HspX) of Mtb, a well-established biomarker of this pathogen, directly in pre-treated sputum samples. The method relies on highly specific monoclonal antibodies which are previously immobilized on the plasmonic sensor surface. This technology allows the direct detection of the biomarker without amplification steps, showing a Limit of Detection (LOD) of 0.63 ng mL-1 and a Limit of Quantification (LOQ) of 2.12 ng mL-1. The direct analysis in pre-treated sputum shows significant differences in the HspX concentration in patients with tuberculosis (with concentration levels in the order of 116-175 ng mL-1) compared with non-tuberculosis infected patients (values below the LOQ of the assay).
关键词： SPR Immunosensor,Tuberculosis diagnosis,HspX recombinant protein,human sputum samples,Point-of-Care device
更新于2025-09-19 17:13:59