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Ultrafast Protein Response in Channelrhodopsin-2 Studied by Time-Resolved IR Spectroscopy
摘要： Ultrafast IR transient absorption in the carbonyl vibrational region of protonated aspartate and glutamate residues in Channelrhodopsin-2 from Chlamydomonas reinhardtii shows immediate protein response to retinal excitation. The observed difference bands are formed directly after the excitation on the sub-ps time scale and were assigned to side chains in the retinal vicinity, such as D156 and E90. This finding implies an ultrafast and effective energy transfer from the retinal to its environment via hydrogen-bonded networks and reveals extraordinary strong chromophore-protein coupling and intense interaction within the protein. Relevance on the protein function as optically gated ion channel are discussed.
关键词： Channelrhodopsin-2,protein response,ion channel,Ultrafast IR transient absorption,hydrogen-bonded networks,chromophore-protein coupling,retinal excitation
更新于2025-09-23 15:19:57
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Visible-Light-Activated High-Density Materials for Controlled in Vivo Insulin Release
摘要： In this work, we describe the synthesis, characterization, and ultimate in vivo assessment of second-generation insulin photoactivated depot (PAD) materials. These are the first to use visible light to stimulate insulin release and have an in vivo performance that is 28-fold improved relative to first-generation materials. This improvement is due to two major factors linked to the utilized chemistry: (1) we have incorporated the coumarin photo-cleavable group, which increases the photorelease wavelength into the visible range, enhancing tissue penetration of the light; (2) photo-toggling of insulin solubility is produced by linking three insulin molecules to a central bridge via light cleaved groups, and not by bonding to a large polymer. The resulting trimer is, therefore, highly dense (87% insulin dry w/w) but retains the insolubility required of the approach. Only after irradiation with visible light is native, soluble insulin released from the dermal depot. This high density increases the amount and ease of insulin release, as the density of photolytic groups is 10?20-fold higher than in polymer-based first-generation materials. We have synthesized new azide-terminated coumarin linkers that we react with the amine groups of insulin. Using mass spectrometry methods, we identify the sites of reaction and purify individual isomers, which we demonstrate have in vitro photolysis rates that are within a factor of 2 of each other. We then reacted these terminal azide groups with a tridentate strained alkyne linker. We show that the resulting insulin trimer is highly insoluble, but can be milled into injectable particles that release insulin only in response to light from a 406 nm light source. Finally, we demonstrate that these materials have a significantly improved in vivo performance, releasing 28-fold more insulin on a per energy basis than first-generation materials.
关键词： photolysis,protein chemistry,insulin,coumarin,click chemistry
更新于2025-09-23 15:19:57
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Protein Content Sensor based on Plastic Optical Fiber
摘要： Plastic optical fiber (POF) content protein sensor has been developed. This sensor is made with a gamma and spiral configuration, with a cladding peel length of 5 cm. POF sensor is dipped into the container containing Bovine Serum Albumin (BSA) solution with different concentrations of 0.1-1 mg/ml. Both ends of the sensor are connected by Light Emiting Diode (LED) and Phototransistor. The LED light will propagate through the POF and it is received by the phototransistor. Light that propagates will be interfenced due to increasing of protein concentration around the sensor, so that the output voltage will be affected. The output voltage will be forwarded to Arduino Uno and read by the computer. The best results were obtained in a spiral configuration with 3 bending with a sensitivity of 0.007 Vml/mg and a resolution of 0.142 mg/ml. This method has advantages that are easy fabrication, simple measurement process, and low cost.
关键词： protein sensor,Plastic optical fiber,Phototransistor,Arduino Uno,Bovine Serum Albumin,LED
更新于2025-09-23 15:19:57
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A UV cross-linking method combined with infrared imaging to analyse RNA–protein interactions
摘要： Photo cross-linking of proteins with short RNA oligomers is a classical method to study RNA–protein interactions that are implicated in many aspects of RNA metabolism and function. Most commonly, this involves the use of [c-32P]-labeled RNA probes. Although very sensitive, these procedures are complicated by the safety issues associated with the use of radioisotopes. Here, we describe a modi?ed UV cross-linking method using oligonucleotide probes end labelled with the infrared dye IRDyeVR 800. After UV cross-linking, proteins are separated by SDS-PAGE and cross-linked products are visualized with the OdysseyVR Infrared Imaging system. This end labelling approach provides a streamlined alternative to random labelling which reduces the ef?ciency of in-vitro transcription. End labelling is also independent of the length of the probe, thus facili- tating quantitative comparisons. To validate the method, we have con?rmed the binding of HuD to the 30-UTR of the mRNA for the microtubule-associated protein tau, implicated in the pathogenesis of Alzheimer’s disease. UV cross-linking of HuD with a labeled 21-mer probe was successfully performed using a recombinant puri?ed glutathione-S-transferase–HuD fu- sion protein as well as with lysates from CHO cells transfected with HuD cDNA. UV cross-linking combined with infrared imaging offers a convenient and robust strategy to analyse RNA–protein interactions and their emerging importance in disease.
关键词： Tau,RNA-binding protein,UV cross-linking,neurodegeneration,HuD,OdysseyVR
更新于2025-09-23 15:19:57
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Synthesis and photophysics of benzazole based triazoles with amino acid-derived pendant units. Multiparametric optical sensors for BSA and CT-DNA in solution
摘要： Herein we report the synthesis of a series of amino acid-derived triazoles by an organocatalytic cycloaddition reaction between azides and carbonyl compounds, catalyzed by a simple amine. These compounds present absorption maxima located in the UV-B ascribed to fully spin and symmetry allowed electronic transitions and a main fluorescence emission in the UV-A (~380 nm) with a relatively large Stokes shift (5700 cm-1). No significant solvatochromism was observed in both ground and excited states. Unexpectedly, the benzoxazole derivatives presented much higher fluorescence quantum yield values (40-80%) of compared to the sulfur analogues (3-6%). In addition, the DNA binding assays indicated that these compounds presented strong interaction with CT-DNA, which could be attributed to π-stacking and intermolecular hydrogen-bonding. The interaction of the benzazoles with bovine serum albumin (BSA) was also investigated, where a suppression mechanism was observed. In each case, docking was performed to better understand the observed interactions.
关键词： fluorescent sensor,DNA intercalation,protein detection,amino acid-derived triazole,docking studies
更新于2025-09-23 15:19:57
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Use of BODIPY-Labeled ATP Analogues in the Development and Validation of a Fluorescence Polarization-Based Assay for Screening of Kinase Inhibitors
摘要： The screening of compound libraries to identify small-molecule modulators of specific biological targets is crucial in the process for the discovery of novel therapeutics and molecular probes. Considering the need for simple single-tool assay technologies with which one could monitor “all” kinases, we developed a fluorescence polarization (FP)-based assay to monitor the binding capabilities of protein kinases to ATP. We used BODIPY ATP-y-S as a probe to measure the shift in the polarization of a light beam when passed through the sample. We were able to optimize the assay using commercial Protein Kinase A (PKA) and H7 efficiently inhibited the binding of the probe when added to the reaction. Furthermore, we were able to employ the assay in a high-throughput fashion and validate the screening of a set of small molecules predicted to dock into the ATP-binding site of PKA. This will be useful to screen larger libraries of compounds that may target protein kinases by blocking ATP binding.
关键词： high-throughput screening,ATP-binding site,protein kinases,fluorescence polarization,BODIPY ATP-y-S
更新于2025-09-23 15:19:57
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Effect of low power laser irradiation on bio-physical properties of wheat seeds
摘要： The production of quality food is indispensable and essential to overcome its global shortage. In order to exercise the facile route to acquire massive production, a laser irradiation treatment method was established. We have investigated the effects of diode laser irradiation on wheat seeds germination, its early growth and biochemical parameters. The variation in germination parameters, caused by laser treatment were monitored carefully. A comparison study was accomplished to examine the laser irradiation effects on treated and control seeds. The red laser indicated an enhancement of 25% in germination percentage, 4.68 cm in shoot length, and 0.64 cm in root length while green laser showed 6.25% less germination percentage than the untreated seeds. Further green laser showed 9.67 cm increase in shoot length, 5.39 cm in root length as compared to untreated seeds. It was found that the seeds which were irradiated with green laser showed an amount of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), of 11.9, 10, and 1.53 per mg of protein, respectively as compared to control seeds. Thus, these investigations are promising and suitable for upcoming research on a large scale.
关键词： Wheat,Biochemical parameters,Protein,Germination parameter,Diode laser
更新于2025-09-23 15:19:57
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Infrared spectroscopic analysis on structural changes around the protonated Schiff base upon retinal isomerization in light-driven sodium pump KR2
摘要： Krokinobacter rhodopsin 2 (KR2) was discovered as the first light-driven sodium pumping rhodopsin (NaR) in 2013, which contains unique amino acid residues on C-helix (N112, D116, and Q123), referred to as an NDQ motif. Based on the recent X-ray crystal structures of KR2, the sodium transport pathway has been investigated by various methods. However, due to complicated structural information around the protonated Schiff base (PRSB) region in the dark state and lack of structural information in the intermediates with sodium bound in KR2, detailed sodium pump mechanism is still unclear. Here we applied comprehensive low-temperature light-induced difference FTIR spectroscopy on isotopically labeled KR2 WT and site-directed mutant proteins (N112A, D116E, R109A, and R109K). We assigned the N-D stretching vibration of the PRSB at 2095 cm?1 and elucidate the hydrogen bonding interaction with D116 (a counter ion for the PRSB). We also assigned strongly hydrogen-bonded water (2333 cm?1) near R109 and D251, and found that presence of a positive charge at the position of R109 is prerequisite for the pumping function of KR2.
关键词： Retinal,Sodium ion pump,Low-temperature FTIR,Protein-bound water,Hydrogen bond
更新于2025-09-23 15:19:57
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Cancer cella??specific protein delivery by optoporation with lasera??irradiated gold nanorods
摘要： The delivery of macromolecules into living cells is challenging since in most cases molecules are endocytosed and remain in the endo-lysosomal pathway where they are degraded before reaching their target. Here, a method is presented to selectively improve cell membrane permeability by nanosecond laser irradiation of gold nanorods (GNRs) with visible or near-infrared irradiation in order to deliver proteins across the plasma membrane, avoiding the endo lysosomal pathway. GNRs were labelled with the anti-EGFR (epidermal growth factor receptor) antibody Erbitux to target human ovarian carcinoma cells OVCAR-3. Irradiation with nanosecond laser pulses at wavelengths of 532 nm or 730 nm is used for transient permeabilization of the cell membranes. As a result of the irradiation, the uptake of an anti-Ki-67 antibody was observed in about 50 % of the cells. The results of fluorescence lifetime imaging show that the GNR detached from the membrane after irradiation.
关键词： gold nanorods,optical window,protein delivery,near infrared radiation,nanosecond pulsed laser
更新于2025-09-23 15:19:57
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PACAP through EGFR transactivation preserves human corneal endothelial integrity
摘要： The corneal endothelium is composed of a single hexagonal‐shaped cells layer adherent to the Descemet's membrane. The primary function of these cells is maintaining of tissue clarity by regulating its hydration. Trauma, aging or other pathologies cause their loss, counterbalanced by enlargement of survived cells fluid pumping to and from the stroma. unable to guarantee an efficient Regenerative medicine using human corneal endothelial cells (HCECs) isolated from peripheral corneal‐scleral tissue of a donor could be an attractive solution, overcoming transplantation problems. In a previous study, we have demonstrated that HCECs treatment with pituitary adenylate cyclase–activating polypeptide following growth factors deprivation prevents their degeneration. (PACAP) However, the molecular mechanism mediating this effect has not been clarified, yet. Here, we have shown for the first time the expression of PACAP and its receptor (PAC1R) in human corneal endothelium and demonstrated that this peptide, selectively binding to PAC1R, induces epidermal growth factor receptor (EGFR) phosphorylation and the MAPK/ERK1/2 signaling pathway activation. In conclusion, our data have suggested that PACAP could represent an important trophic factor in maintaining human corneal endothelial integrity through EGFR transactivation. Therefore, PACAP, as well as epidermal growth factor and fibroblast growth factor, could co‐operate to guarantee tissue physiological functioning by supporting corneal endothelial barrier integrity.
关键词： epidermal growth factor receptor,human corneal endothelial cells,mitogen‐activated protein kinase,pituitary adenylate cyclase–activating polypeptide
更新于2025-09-23 15:19:57