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Surface Plasmon Resonance Based Recent Advances in Understanding Plant Development and Related Processes
摘要： Since its introduction in the early 1990s, SPR has now become a powerful research tool for studying specificity, affinity and real time kinetics of a broad range of biomolecular interactions, including protein-DNA, protein-protein, protein-carbohydrate, protein-RNA and protein-lipid interactions. Surface plasmon resonance (SPR) has provided crucial information on the mechanisms of molecular interactions accompanying varied aspects of plant development. The structure-function relationship of various lectins depends on the quaternary arrangement of its monomers. Novel findings have been made in plant hormone research using SPR as a technique. Thus, new salicylic acid binding proteins (SABPs) have been identified in Arabidopsis. These include α-ketoglutarate dehydrogenase E2 subunit, glutathione S-transferases, the oligopeptidases TOP2 and TOP1, and members of GAPDH protein family. By immobilizing biotin-labelled DELLA peptides on the sensor chip and AtGID1a [Arabidopsis gibberellic acid (GA) receptor] as analyte, GA4 has been observed to maximally enhance binding between DELLA and GID1. Molecularly imprinted monolayer (MIM)-decorated SPR detection method precisely differentiates between similar plant hormones, such as, IAA, 1H-indole-3-butyric acid (IBA) and kinetin (KT), with detection limits around sub-picomolar range. Coronatine Insensitive-1 (COI1) has been shown to act as a jasmonic acid receptor using SPR. Ricin, a plant toxin, was detected at a concentration 2,500 times less than the minimum lethal dose (200 ng.ml-1) using a SPR biosensor. Real time binding kinetic studies of viral proteins (VirE1 and VirE2) and ssDNA using SPR have shown that their binding is strongly influenced by substrate and it occurs at poly T sequences and not at polyA and dsDNA. An interaction between the replicase protein (p93) of cucumber necrosis tombusvirus (CNV) with the host protein, Hsp 70 (molecular chaperone), has revealed the potential role of Hsp90 in the assembly of viral replicase. SPR analysis from a small library of phytochemicals has shown ellagitannin geraniin as one of the most potent inhibitor of Hsp90 (a stabilizer of many oncoproteins). Future applications of SPR technique are likely to provide tremendous inputs into the molecular understanding of plant development and related processes.
关键词： Protein-chaperone interactions,Protein-carbohydrate interactions,Plant viruses,Surface plasmon resonance,Phytohormones,Protein-nucelic acid interactions,Xenobiotics,Protein-protein interactions
更新于2025-09-23 15:21:01
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Protein Corona Formation of Human Serum Albumin with Carbon Quantum Dots from Roasted Salmon
摘要： As the food-borne nanoparticles enter the biological system, they will contact with various proteins to form protein coronas, which can affect their physicochemical properties and biological identity. In this study, the protein corona formation of carbon quantum dots (CQDs) from roast salmon with human serum albumin (HSA), as well as biological identity involving cell apoptosis, energy, glucose and lipid metabolism and acute toxicity in mice, were investigated. The HSA-CQD coronas formed between HSA and CQDs via static binding mechanism, and the binding site of CQDs on HSA located both in Sudlow’s site I and site II. The HSA-CQD coronas entered the cytoplasm and present in lysosomes or autolysosomes. Importantly, the HSA coronas mitigated the cytotoxicity of CQDs from 18.65% to 9.26%, and the energy metabolism was rectified from glycolytic to aerobic metabolism. The glucose and lipid metabolite profile of the HSA-CQD coronas differed from that of the CQDs, indicating that HSA-CQD coronas rectified disturbance in metabolism. Histopathological and blood biochemical analysis revealed no statistically significant difference between the testing and control mice after a single CQDs dose of 2000 mg/kg body weight. Overall, the results confirmed the formation of protein corona between HSA and food-borne fluorescent CQDs, and could be helpful for evaluating the safety of fluorescent CQDs from roast food items.
关键词： cytotoxicity,acute toxicity,protein coronas,human serum albumin,carbon quantum dots,metabolism,food-borne nanoparticles
更新于2025-09-23 15:21:01
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Picomolar Biosensing and Conformational Analysis Using Artificial Bidomain Proteins and Terbiuma??Toa??Quantum Dot F??rster Resonance Energy Transfer
摘要： Although antibodies remain a primary recognition element in all forms of biosensing, functional limitations arising from their size, stability, and structure have motivated the development and production of many different artificial scaffold proteins for biological recognition. However, implementing such artificial binders into functional high-performance biosensors remains a challenging task. Here, we present the design and application of F?rster resonance energy transfer (FRET) nanoprobes comprised of small artificial proteins (αRep bidomains) labeled with a Tb complex (Tb) donor on the C-terminus and a semiconductor quantum dot (QD) acceptor on the N-terminus. Specific binding of one or two protein targets to the αReps induced a conformational change that could be detected by time-resolved Tb-to-QD FRET. These single-probe FRET-switches were used in a separation-free solution-phase assay to quantify different protein targets at subnanomolar concentrations and measure the conformational changes with subnanometer resolution. Probing ligand-receptor binding under physiological conditions at very low concentrations in solution is a special feature of FRET that can be efficiently combined with other structural characterization methods to develop, understand, and optimize artificial biosensors. Our results suggest that the αRep FRET nanoprobes have a strong potential for their application in advanced diagnostics and intracellular live cell imaging of ligand-receptor interactions.
关键词： protein structure,molecular ruler,lanthanides,molecular diagnostics,FRET,binding assay
更新于2025-09-23 15:21:01
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Mutated Human P-Selectin Glycoprotein Ligand-1 and Viral Protein-1 of Enterovirus 71 Interactions on Au Nanoplasmonic Substrate for Specific Recognition by Surface-Enhanced Raman Spectroscopy
摘要： Protein tyrosine sulfation is a common post-translational modification that stimulates intercellular or extracellular protein-protein interactions and is responsible for various important biological processes, including coagulation, inflammation, and virus infections. Recently, human P-selectin glycoprotein ligand-1 (PSGL-1) has been shown to serve as a functional receptor for enterovirus 71 (EV71). It has been proposed that the capsid viral protein VP1 of EV71 is directly involved in this specific interaction with sulfated or mutated PSGL-1. Surface-enhanced Raman spectroscopy (SERS) is used to distinguish PSGL-1 and VP1 interactions on an Au nanoporous substrate and identify specific VP1 interaction positions of tyrosine residue sites (46, 48, and 51). The three tyrosine sites in PSGL-1 were replaced by phenylalanine (F), as determined using SERS. A strong phenylalanine SERS signal was obtained in three regions of the mutated protein on the nanoporous substrate. The mutated protein positions at (51F) and (48F, 51F) produced a strong SERS peak at 1599–1666 cm?1, which could be related to a binding with the mutated protein and anti-sulfotyrosine interactions on the nanoporous substrate. A strong SERS effect of the mutated protein and VP1 interactions appeared at (48F), (51F), and (46F, 48F). In these positions, there was less interaction with VP1, as indicated by a strong phenylalanine signal from the mutated protein.
关键词： viral protein 1,P-selectin glycoprotein ligand-1,surface-enhanced Raman spectroscopy,nanoporous,phenylalanine
更新于2025-09-23 15:21:01
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Ultrafast Laser Processing of Nanostructured Patterns for the Control of Cell Adhesion and Migration on Titanium Alloy
摘要： Femtosecond laser texturing is a promising surface functionalization technology to improve the integration and durability of dental and orthopedic implants. Four different surface topographies were obtained on titanium-6aluminum-4vanadium plates by varying laser processing parameters and strategies: surfaces presenting nanostructures such as laser-induced periodic surface structures (LIPSS) and ‘spikes’, associated or not with more complex multiscale geometries combining micro-pits, nanostructures and stretches of polished areas. After sterilization by heat treatment, LIPSS and spikes were characterized to be highly hydrophobic, whereas the original polished surfaces remained hydrophilic. Human mesenchymal stem cells (hMSCs) grown on simple nanostructured surfaces were found to spread less with an increased motility (velocity, acceleration, tortuosity), while on the complex surfaces, hMSCs decreased their migration when approaching the micro-pits and preferentially positioned their nucleus inside them. Moreover, focal adhesions of hMSCs were notably located on polished zones rather than on neighboring nanostructured areas where the protein adsorption was lower. All these observations indicated that hMSCs were spatially controlled and mechanically strained by the laser-induced topographies. The nanoscale structures influence surface wettability and protein adsorption and thus influence focal adhesions formation and finally induce shape-based mechanical constraints on cells, known to promote osteogenic differentiation.
关键词： cell adhesion,multiscale-patterning,wettability,human mesenchymal stem cell,femtosecond laser,cell spreading,cell motility,protein adsorption
更新于2025-09-23 15:21:01
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Structural dynamics in proteins induced by and probed with X-ray free-electron laser pulses
摘要： X-ray free-electron lasers (XFELs) enable crystallographic structure determination beyond the limitations imposed upon synchrotron measurements by radiation damage. The need for very short XFEL pulses is relieved through gating of Bragg diffraction by loss of crystalline order as damage progresses, but not if ionization events are spatially non-uniform due to underlying elemental distributions, as in biological samples. Indeed, correlated movements of iron and sulfur ions were observed in XFEL-irradiated ferredoxin microcrystals using unusually long pulses of 80 fs. Here, we report a femtosecond time-resolved X-ray pump/X-ray probe experiment on protein nanocrystals. We observe changes in the protein backbone and aromatic residues as well as disulfide bridges. Simulations show that the latter’s correlated structural dynamics are much slower than expected for the predicted high atomic charge states due to significant impact of ion caging and plasma electron screening. This indicates that dense-environment effects can strongly affect local radiation damage-induced structural dynamics.
关键词： plasma electron screening,radiation damage,ion caging,structural dynamics,protein nanocrystals,X-ray free-electron lasers
更新于2025-09-23 15:21:01
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A Recombinant Fluorescent Peptidomimetic Tracer for Immunodetection of Imidaclothiz
摘要： Peptidomimetic and anti-immunocomplex peptides, which could be readily isolated from phage display library, have shown a great potential for small molecule immunoassay development because they typically improve the sensitivity and avoid the use of chemical haptens as coating or tracers antigens. However, the phage borne peptides are unconventional immunoassay reagent, which greatly limits their use in commercial applications and requires secondary reagents for detection. In order to overcome these limitations, we used C2-15, a peptidomimetic of imidaclothiz, as a model peptide fused to emerald green fluorescent protein (EmGFP) at the N terminus (C2-15-EmGFP) and C terminus (EmGFP-C2-15) to generate novel fluorescent peptide tracers. Both recombinant fluorophores reacted with similar affinity to the anti-imidaclothiz monoclonal antibody 1E7, but due to its higher expression C2-15-EmGFP was chosen to develop a competitive magnetic separation fluorescence immunoassay (MSFIA). After a competitive step with the analyte, the C2-15-EmGFP/antibody complex bound to the magnetic beads was separated with a magnet, and due to the fast dissociation of the peptide-antibody interaction, the fluorescence signal was detected following the spontaneous dissociation of the complex in fresh buffer. The concentration of imidaclothiz causing the 50% inhibitory concentration (IC50) was 11.00 ng mL-1, and the MSFIA performed with excellent recovery and good correlation with high-performance liquid chromatography in different matrices.
关键词： Magnetic nanoparticles,Emerald green fluorescent protein,Recombinant peptide,Imidaclothiz,Peptidomimetic
更新于2025-09-23 15:21:01
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A genetically encoded photosensitizer protein facilitates the rational design of a miniature photocatalytic CO2-reducing enzyme
摘要： Photosensitizers, which harness light energy to upgrade weak reductants to strong reductants, are pivotal components of the natural and artificial photosynthesis machineries. However, it has proved difficult to enhance and expand their functions through genetic engineering. Here we report a genetically encoded, 27 kDa photosensitizer protein (PSP), which facilitates the rational design of miniature photocatalytic CO2-reducing enzymes. Visible light drives PSP efficiently into a long-lived triplet excited state (PSP*), which reacts rapidly with reduced nicotinamide adenine dinucleotide to generate a super-reducing radical (PSP?), which is strong enough to reduce many CO2-reducing catalysts. We determined the three-dimensional structure of PSP? at 1.8 ? resolution by X-ray crystallography. Genetic engineering enabled the site-specific attachment of a nickel–terpyridine complex and the modular optimization of the photochemical properties of PSP, the chromophore/catalytic centre distance and the catalytic centre microenvironment, which culminated in a miniature photocatalytic CO2-reducing enzyme that has a CO2/CO conversion quantum efficiency of 2.6%.
关键词： quantum efficiency,photosensitizer protein,visible light,photocatalytic CO2-reducing enzymes,X-ray crystallography,nickel–terpyridine complex,genetic engineering
更新于2025-09-23 15:21:01
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Alkyne Functionalization of a Photoactivated Ruthenium Polypyridyl Complex for Click-Enabled Serum Albumin Interaction Studies
摘要： Studying metal-protein interactions is key for understanding the fate of metallodrugs in biological systems. When a metal complex is not emissive and too weakly bound for mass spectrometry analysis, however, it may become challenging to study such interactions. In this work a synthetic procedure was developed for the alkyne functionalization of a photolabile ruthenium polypyridyl complex, [Ru(tpy)(bpy)(Hmte)](PF6)2, where tpy = 2,2′:6′,2′′-terpyridine, bpy = 2,2′-bipyridine, and Hmte = 2-(methylthio)ethanol. In the functionalized complex [Ru(HCC-tpy)(bpy)(Hmte)](PF6)2, where HCC-tpy = 4′-ethynyl-2,2′:6′,2′′-terpyridine, the alkyne group can be used for bioorthogonal ligation to an azide-labeled fluorophore using copper-catalyzed “click” chemistry. We developed a gel-based click chemistry method to study the interaction between this ruthenium complex and bovine serum albumin (BSA). Our results demonstrate that visualization of the interaction between the metal complex and the protein is possible, even when this interaction is too weak to be studied by conventional means such as UV?vis spectroscopy or ESI mass spectrometry. In addition, the weak metal complex-protein interaction is controlled by visible light irradiation, i.e., the complex and the protein do not interact in the dark, but they do interact via weak van der Waals interactions after light activation of the complex, which triggers photosubstitution of the Hmte ligand.
关键词： ruthenium polypyridyl complex,click chemistry,bovine serum albumin,photoactivation,alkyne functionalization,metal-protein interactions
更新于2025-09-23 15:21:01
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Drug Delivery System Using Photothermal Effect of Gold Nanorods
摘要： Gold nanorods have absorption bands in the near-infrared region. The absorbed light energy is converted into heat by gold nanorods. Therefore, drug delivery systems that can be controlled by the heat produced by near-infrared light irradiation will be constructed. First, we modified gold nanorods with double-stranded DNA. That is, release of single stranded DNA is induced by the produced heat from the gold nanorods. We also constructed a controlled release system of PEG chain mediated by retro Diels-Alder reaction induced by the photothermal effect. Next, we designed transdermal protein delivery system enhanced by the photothermal effect. We first casted gold nanorods, acting as a heating device in response to near-infrared light irradiation, onto the skin surface. After applying an aqueous solution of ovalbumin to the skin, the skin was irradiated by near-infrared laser light. The irradiation increased the skin temperature resulting in an efficient translocation of ovalbumin into the skin. Thus, the controlled-release systems and enhanced transdermal protein delivery system triggered by near-infrared light irradiation will be further expanded to development of functional drug delivery system.
关键词： photothermal effect,gold nanorods,transdermal protein delivery,drug delivery system,near-infrared light
更新于2025-09-23 15:21:01