Protein Fluorescence Research Articles

Molecular Insights into the Conformational and Binding Behaviors of Human Serum Albumin Induced by Surface-Active Ionic Liquids.

Extensive research has been carried out to investigate the stability and function of human serum albumin (HSA) when exposed to surface-active ionic liquids (SAILs) with different head groups (imidazolium, morpholinium, and pyridinium) and alkyl chain lengths (ranging from decyl to tetradecyl). Analysis of the protein fluorescence spectra indicates noticeable changes in the secondary structure of HSA with varying concentrations of all SAILs tested. Helicity calculations based on the Fourier transform infrared (FTIR) data show that HSA becomes more organized at the micellar concentration of SAILs, leading to an increased protein activity at this level. Small-angle neutron scattering (SANS) data confirm the formation of a bead-necklace structure between the SAILs and HSA. Atomistic molecular dynamics (MD) simulation results identify several hotspots on the protein surface for interaction with SAIL, which results in the modulation of protein conformational fluctuation and stability. Furthermore, fluorescence resonance energy transfer (FRET) experiments with the intramolecular charge transfer (ICT) probe trans-ethyl p-(dimethylamino) cinnamate (EDAC) demonstrate that higher alkyl chain lengths and SAIL concentrations result in a significantly increased energy transfer efficiency. The findings of this study provide a detailed molecular-level understanding of how the protein structure and function are affected by the presence of SAILs, with potential implications for a wide range of applications involving protein-SAIL composite systems.

A novel heat-inducible plasmid-driven T7 system for enhancing carbonic anhydrase in engineered Escherichia coli strains

Over the past decades, the heat-inducible (HI) promoter, derived from cI857 phage system has been widely utilized. However, the developments of alternative temperature-controlled promoters are limited. In this study, we explored a novel HI promoter through PCR amplification using the degenerate primer design. BLAST analysis identified this sequence as a partial plsB on Escherichia coli chromosome. Antisense RNAs targeting distinct regions of HI promoter were applied to examine the mechanism. The fluorescence of super-folder green fluorescent protein (sfGFP) driven by the HI promoter showed 5.5-fold increase from 30 to 42 °C, whereas the truncated HI yielded a trace amount of sfGFP. Among various E. coli strains, BL21 exhibited the highest fluorescence, reaching 3976 a.u. at 42 °C. Subsequently, the novel HI promoter was employed to drive T7RNA polymerase within a plasmid-driven T7 (PDT7) plasmid, serving its capability to express sfGFP and carbonic anhydrases (CA), respectively. The maximum intensity of sfGFP reached 38,702 a.u., and CA activity surged to 14286 WAU/mL in W3110 among other strains. Finally, the highest CA activity was 27,521 WAU/mL at pH 9. The promising results from the novel heat-inducible promoter-driven T7RNAP, incorporating the T7 terminator as HI-PDT7-TT, demonstrate potential for expressing more heterogeneous proteins across various chassis in the future.

Possibility of using the imaged capillary isoelectric method as a multi-attribute method for bispecific antibodies.

An imaged capillary isoelectric focusing (icIEF)-based method was developed and validated as a multi-attribute method for a bispecific antibody (BsAb). First, as the traditional application of the icIEF method, it serves as an identity assay and purity assay for the BsAb. Second, the method can also be used as an impurity assay for the homodimer monoclonal antibodies generated during BsAb assembly. The homodimer impurity analysis for BsAb is usually done by hydrophobic interaction chromatography methods in the industry. The icIEF method has good sensitivity (down to 4µg/mL in a limit of quantitation) when UV fluorescence detection is used, which detects the native fluorescence of proteins. This is the first report that an icIEF method has been applied as impurity assay.

Characterization and classification of pathogenic bacteria using native fluorescence and spectral deconvolution.

Identification and classification of pathogenic bacterial strains is of current interest for the early treatment of diseases. In this work, protein fluorescence from eight different pathogenic bacterial strains were characterized using steady state and time resolved fluorescence spectroscopy. The spectral deconvolution method was also employed to decompose the emission contribution from different intrinsic fluorophores and extracted various key parameters, such as intensity, emission maxima, emission line width of the fluorophores, and optical redox ratio. The change in average lifetime values across different bacterial strains exhibits good statistical significance (p ≤ 0.01). The variations in the photophysical characteristics of bacterial strains are due to the different conformational states of the proteins. The stepwise multiple linear discriminate analysis of fluorescence emission spectra at 280 nm excitation across eight different bacterial strains classifies the original groups and cross validated group with 100% and 99.5% accuracy, respectively.

Head-to-head Comparison of Green Fluorescent Protein (GFP) Imaging With Luciferase-luciferin Imaging In Vivo Using Single-nanometer Laser-excitation Tuning and an Ultra-low-light-detection Camera and Optics Demonstrates the Superiority of GFP.

Genetic reporters encoding fluorescent proteins or luciferase have been used in vivo for the last three decades with claims about their superiority or inferiority over each other. In the present report, a head-to-head in vivo comparison of green fluorescent protein (GFP) fluorescence imaging and luciferase-luciferin imaging, using single-nanometer laser-excitation tuning of fluorescence excitation and an ultra-low-light-detection camera and optics was performed. Mouse Lewis-lung carcinoma cells labeled with GFP (LLC-GFP) or luciferase (LL/2-Luc2) were injected subcutaneously into the flank of nude mice. One week after injection, GFP-fluorescence imaging and luciferase-luciferin imaging was performed using the UVP Biospectrum Advanced system with excitation at 487 nm and peak emission at 513 nm for GFP, and with emission at 560 nm for luciferase-luciferin. GFP fluorescence images were obtained at 0, 10, and 20 min. Luciferase-luciferin images were obtained 10 and 20 min after the injection of D-luciferin. The intensity of GFP images was 55,909 at 0 min, 56,186 at 10 min, and 57,085 at 20 min, and maintained after 20 min. The intensity of luciferase-luciferin images was 28,065 at 10 min after the injection of D-luciferin and 5,199 at 20 min after the injection. The intensity of luciferase-luciferin images decreased by approximately 80% at 20 min compared to 10 min. An exposure time of 30 s for luciferase-luciferin imaging was needed compared to 100 ms for GFP fluorescence imaging in order to detect signals. An imaging system with single-nanometer tuning fluorescence excitation and an ultra-low-light detection camera and optics was able to directly visualize both GFP and luciferase-luciferin images in vivo. The intensity and stability of the signals were both greater for GFP than for luciferase-luciferin, and the exposure time for GFP was 300 times faster, demonstrating the superiority of GFP.

DNA-controlled protein fluorescence: Design of aptamer-split peptide hetero-modulator for GFP to respond to intracellular ATP levels.

Enabling the precise control of protein functions with artificially programmed reaction patterns is beneficial for investigating biological processes. Although several strategies have been established that employ the programmability of nucleic acid, they have been limited to DNA hybridization without external stimuli or target binding. Here, we report an approach for the DNA-mediated control of the tripartite split-GFP assembly via aptamers with responsiveness to intracellular small molecules as stimuli. We designed a novel structure-switching aptamer-peptide conjugate as a hetero modulator for splitGFP in response to ATP. By conjugating two peptides (S10/11) derived from the tripartite split-GFP to ATP aptamer, we achieved GFP reassembly using only ATP as a trigger molecule. The response to ATP at≥4 mM concentrations indicated that it can be applied to respond to intracellular ATP in live cells. Furthermore, our hetero-modulator exhibited high and long-term stability, with a half-life of approximately four days in a serum stability assay, demonstrating resistance to nuclease degradation. We validated that our aptamer-modulator splitGFP was successfully reconstituted in the cell in response to intracellular ATP levels. Our aptamer-modulated splitGFP platform can be utilized to monitor a wide range of intracellular metabolites by replacing the aptamer sequence.

A thiol-anchored solvatochromic and fluorogenic molecular rotor for covalent protein labeling in SDS-PAGE and mitochondria specific fluorescence imaging.

Fluorescent labeling is a widely used method for protein detection and fluorescence imaging. A solvatochromic and fluorogenic molecular rotor DASPBCl was developed for covalent protein labeling in solution and SDS-PAGE, and also for stable mitochondria labeling and fluorescence imaging. The dye DASPBCl consisted of a 4-(N,N-dimethylamino)phenyl moiety as the electron donor and a positively charged N-benzylpyridinium moiety as the electron acceptor. A benzyl chloride group was introduced into the pyridine moiety for covalent labeling of thiol in proteins. When the fluorescent dye DASPBCl is covalently labeled to the thiol of proteins, significantly enhanced fluorescence was obtained, which is attributed to the polarity sensitivity caused solvatochromic effect from the hydrophobic protein structure and the viscosity sensitivity caused fluorogenic effect from the restriction of single bond rotation. DASPBCl exhibits high sensitivity and good linear response for protein detection in SDS-PAGE analysis with both the pre-staining method and post-staining method. DASPBCl was also successfully used for covalently protein-anchored fluorescence imaging of mitochondria in living cells.

Design and investigation of celastrol‐peptide nanoassemblies and their binding interactions with superoxide dismutase 1 and its mutants

AbstractThe misfolding and aggregation of superoxide dismutase 1 (SOD1) and its mutants has been implicated in amyotrophic lateral sclerosis (ALS). In this study, we have created three peptide conjugates with the antioxidant pentacyclic terpene celastrol and examined their interactions with SOD1 and its mutants A4V and G93A. The peptides YYIVS, MPDAHL, and GSGGL are derived from natural sources and are known for their inherent antioxidant properties. Docking studies revealed that most conjugates showed strong binding with the metal binding and electrostatic loops as well as the β1, β5, and β6 hydrophobic core of SOD1. The conjugates were synthesized and self‐assembled into nanoassemblies. Surface plasmon resonance studies further confirmed the binding interactions of the nanoassemblies with the SOD1 proteins. The nanoassemblies were found to internalize into HEK293T cells. The HEK 293T cells were then transfected with GFP fused WT (Wild Type), A4V and G93A SOD1 mutants. Flow cytometry revealed that treatment with celastrol‐peptide nanoassemblies, affected the fluorescence of the SOD1 protein, implying their role in modulating SOD1, particularly for the mutants. N–Acetyl–Leu–Leu–Norleucinal (ALLN) induced SOD1 aggregation was also affected upon treatment with the nanoassemblies. These results suggest that the nanoassemblies may potentially modulate the activity and structure of SOD1.

Effect of scutellarin on BV-2 microglial-mediated apoptosis in PC12 cells via JAK2/STAT3 signalling pathway

Previous studies have shown that scutellarin inhibits the excessive activation of microglia, reduces neuronal apoptosis, and exerts neuroprotective effects. However, whether scutellarin regulates activated microglia-mediated neuronal apoptosis and its mechanisms remains unclear. This study aimed to investigate whether scutellarin can attenuate PC12 cell apoptosis induced by activated microglia via the JAK2/STAT3 signalling pathway. Microglia were cultured in oxygen–glucose deprivation (OGD) medium, which acted as a conditioning medium (CM) to activate PC12 cells, to investigate the expression of apoptosis and JAK2/STAT3 signalling-related proteins. We observed that PC12 cells apoptosis in CM was significantly increased, the expression and fluorescence intensity of the pro-apoptotic protein Bax and apoptosis-related protein cleaved caspase-3 were increased, and expression of the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) was decreased. Phosphorylation levels and fluorescence intensity of the JAK2/STAT3 signalling pathway-related proteins JAK2 and STAT3 decreased. After treatment with scutellarin, PC12 cells apoptosis as well as cleaved caspase-3 and Bax protein expression and fluorescence intensity decreased. The expression and fluorescence intensity of Bcl-2, phosphorylated JAK2, and STAT3 increased. AG490, a specific inhibitor of the JAK2/STAT3 signalling pathway, was used. Our findings suggest that AG490 attenuates the effects of scutellarin. Our study revealed that scutellarin inhibited OGD-activated microglia-mediated PC12 cells apoptosis which was regulated via the JAK2/STAT3 signalling pathway.

A fluorescence-based sensor for calibrated measurement of protein kinase stability in live cells.

Oncogenic mutations can destabilize signaling proteins, resulting in increased or unregulated activity. Thus, there is considerable interest in mapping the relationship between mutations and the stability of signaling proteins, to better understand the consequences of oncogenic mutations and potentially inform the development of new therapeutics. Here, we develop a tool to study protein-kinase stability in live mammalian cells and the effects of the HSP90 chaperone system on the stability of these kinases. We determine the expression levels of protein kinases by monitoring the fluorescence of fluorescent proteins fused to those kinases, normalized to that of co-expressed reference fluorescent proteins. We used this tool to study the dependence of Src- and Raf-family kinases on the HSP90 system. We demonstrate that this sensor reports on destabilization induced by oncogenic mutations in these kinases. We also show that Src-homology 2 and Src-homology 3 domains, which are required for autoinhibition of Src-family kinases, stabilize these kinase domains in the cell. Our expression-calibrated sensor enables the facile characterization of the effects of mutations and small-molecule drugs on protein-kinase stability.

Effect of different pretreatment methods on the stability of pumpkin seed milk and potential mechanism

Pumpkin seeds represent a valuable source of plant protein and can be utilized in the production of plant-based milks. This study aims to investigate the effects of different pretreatment techniques on the stability of Pumpkin Seed Milk (PSM) and explore potential mechanisms. Raw pumpkin seeds underwent pretreatment through roasting, microwaving, and steaming to prepare PSM. Physiochemical attributes such as composition, storage stability, and particle size of PSM were evaluated. Results indicate that stability significantly improved at roasting temperatures of 160 °C, with the smallest particle size (305 ± 40 nm) and highest stability coefficient (0.710 ± 0.002) observed. Nutrient content in PSM remained largely unaffected at 160 °C. Protein oxidation levels, infrared, and fluorescence spectra analysis revealed that higher temperatures exacerbated the oxidation of pumpkin seed emulsion. Overall, roasting raw pumpkin seeds at 160 °C is suggested to enhance PSM quality while preserving nutrient content.

Human coronavirus 229E inactivation on porous and nonporous materials using dielectric barrier discharge (DBD) plasma.

Viruses can spread through contaminated aerosols and contaminated surface materials, and effective disinfection techniques are essential for virus inactivation. Nonthermal plasma-generated reactive oxygen and nitrogen species can effectively inactivate the coronavirus. We aim to interpret the coronavirus inactivation level and mechanism of surface interaction with materials with and without dielectric barrier discharge (DBD) plasma treatment. Nonthermal plasma, particularly surface-type DBD plasma, can inactivate human coronavirus 229E (HCoV-229E) on porous (paper, wood, mask) and nonporous (plastic, stainless steel, glass, Cu) materials. Virus inactivation was analyzed using a 50% tissue culture infectivity dose (TCID50) using cell line, flow cytometry, and immunofluorescence. Surfaces contaminated with HCoV-229E were treated at different time intervals (0-5 h) with and without plasma exposure (natural decay in ambient air conditions). HCoV-229E persistence conformed to the following order: plastic > cover glass > stainless steel > mask > wood > paper > Cu with and without plasma exposure. HCoV-229E was more stable in plastic, cover glass, and stainless steel in 5 h, and the viable virus titer gradually decreased from its initial log10 order of 6.892 to 1.72, 1.53, and 1.32 TCID50/mL, respectively, under plasma exposure. No virus was observed in Cu after treatment for 5 h. The use of airflow, ambient nitrogen, and argon did not promote virus inactivation. Flow cytometry and immunofluorescence analysis demonstrated a low expression level of spike protein (fluorescence intensity) during plasma treatment and in E and M genes expression compared with the virus control.

Double‐stranded RNA uptake for the control of the maize pathogen Cercospora zeina

AbstractRNA interference (RNAi) using double‐stranded RNA (dsRNA) against fungal pathogens is an emerging field of crop disease control. We aimed to evaluate RNAi against the fungus Cercospora zeina causing grey leaf spot (GLS) disease on maize. Orthologues of Dicer‐like 1, Dicer‐like 2, RNA‐dependent RNA polymerase and two copies of Argonaute were identified in the C. zeina genome and were shown to be expressed in vitro and in planta. Confocal microscopy showed that C. zeina took up exogenously applied dsRNA labelled with fluorescein. GFP‐transgenic C. zeina was treated with GFP‐specific dsRNA, and GFP mRNA expression and protein fluorescence were reduced by 57% and 61%, respectively. A Cz3‐dsRNA targeting C. zeina chitin synthase D (CHSD), phosphatidylserine decarboxylase proenzyme 3 (PSD3) and extracellular protein 2 (ECP2) was constructed. Treatment of C. zeina cultures with the Cz3‐dsRNA reduced CHSD expression by 47% and reduced cell viability by 34%. Maize leaves were inoculated with C. zeina conidia, and Cz3‐dsRNA was applied either with the conidia or 16 h later. GLS disease was significantly reduced compared to the water control for the 16 h post‐inoculation (hpi) treatment with Cz3‐dsRNA, but not for the GFP‐dsRNA specificity control or treatments at 0 hpi. We hypothesized that germination of C. zeina conidia was required for effective dsRNA‐mediated control, and this was borne out by microscopy observations that most of the C. zeina conidia (70%) germinated successfully on the maize leaf surface within 16 hpi. This work lays the groundwork for a dsRNA‐based fungicide against this foliar pathogen.

New Ru(II)-p-cymene compounds bearing indomethacin and indomethacin-pyridineamide ligands: synthesis, characterization, computational studies and investigation of their interactions with the Human Serum Albumin

The research interest in ruthenium(II) organometallic compounds has continually increased due to their promising performance in biological assays targeting diverse medicinal applications. This work reports the interactions of the [Ru(II)-η6-p-cymene] framework with the indomethacin (HL1) drug and with its modified analogue indomethacin-pyridineamide (L2). Two new half-sandwich organometallics, [Ru(η6-p-cymene)(L1)Cl] (1) and [Ru(η6-p-cymene)(L2)Cl2] (2), were successfully synthesized and fully characterized by ESI-MS, NMR, ATR-FTIR and UV/VIS spectroscopy. The chemical and electronic structures were corroborated by DFT computational calculations. Compound 2 was chemically stable, keeping the L2 ligand in the coordination sphere in coordinating solvents, and was investigated for the interaction with the Human Serum Albumin (HSA) protein by fluorescence quenching studies. This compound acted as a quencher to the intrinsic protein fluorescence, mainly through binding-related quenching (static) mechanism, showing high value for the apparent association constant (Ka). The analysis of the thermodynamic parameters revealed spontaneous process, presenting low temperature dependence, with electrostatic forces playing a key role in the HSA-compound 2 adduct binding. Site competitive studies suggested that the organometallic 2 may compete for the binding site of ibuprofen (subdomain IIIA) and also for the binding site of Ru(III) naked ion (subdomain IB or IIA). Molecular docking simulations showed that both Ru(II)-arenes may interact through hydrogen bonding with arginine residues, and exhibit high affinity for the microenvironment located in subdomains IIIA and IB. These findings suggest that the HSA protein might be a potential carrier for these Ru(II)-organometallic compounds in the blood plasma.

Inhibitory activity of luteolin on α‐glucosidase and pancreatic lipase in vitro and in vivo

SummaryObesity is becoming a top global health problem. Inhibition of digestive enzymes is an effective approach in weight management due to the reduction of energy absorption. In this study, the inhibitory effects of luteolin on α‐glucosidase and pancreatic lipase were investigated in vitro and in vivo via enzyme kinetics, fluorescence quenching, molecular docking and rat experiments. The results showed that luteolin inhibited α‐glucosidase and pancreatic lipase in a mixed‐type and uncompetitive manner with IC50 values of 29.31 and 42.74 μg mL−1 respectively. The binding of luteolin with the two enzymes caused the quenching of intrinsic fluorescence of protein. Molecular docking simulation revealed that luteolin could be located in the active pocket of α‐glucosidase and interact with the key amino acid residues. Moreover, a hydrophobic interaction and a van der Waals force were formed between luteolin and the key amino acid residues of pancreatic lipase (His264 and Ser153). In vivo studies further confirmed that luteolin could significantly decrease the absorption of glucose and triacylglyceride in rats by inhibiting α‐glucosidase and pancreatic lipase.

Mechanism for improving the in vitro digestive properties of coconut milk by modifying the structure and properties of coconut proteins with monosodium glutamate

In this paper, the effect of monosodium glutamate (MSG) on coconut protein (CP) solubility, surface hydrophobicity, emulsification activity, ultraviolet spectroscopy and fluorescence spectroscopy was investigated. Meanwhile, the changes in the in vitro digestive properties of coconut milk were also further analyzed. MSG treatment altered the solubility and surface hydrophobicity of CP, thereby improving protein digestibility. Molecular docking showed that CP bound to pepsin and trypsin mainly through hydrogen bonds and salt bridges. And MSG increased the cleavable sites of pepsin and trypsin on CP, thus further improving the protein digestibility. In addition, MSG increased the Na+ concentration in coconut milk, promoted flocculation and aggregation between coconut milk droplets, which prevented the binding of lipase and oil droplets and inhibited lipid digestion. These findings may provide new ideas and insights to improve the digestive properties of plant-based milk.

Enhancing subcellular protein localization mapping analysis using Sc2promap utilizing attention mechanisms

BackgroundAberrant protein localization is a prominent feature in many human diseases and can have detrimental effects on the function of specific tissues and organs. High-throughput technologies, which continue to advance with iterations of automated equipment and the development of bioinformatics, enable the acquisition of large-scale data that are more pattern-rich, allowing for the use of a wider range of methods to extract useful patterns and knowledge from them. MethodsThe proposed sc2promap (Spatial and Channel for SubCellular Protein Localization Mapping) model, designed to proficiently extract meaningful features from a vast repository of single-channel grayscale protein images for the purposes of protein localization analysis and clustering. Sc2promap incorporates a prediction head component enriched with supplementary protein annotations, along with the integration of a spatial-channel attention mechanism within the encoder to enables the generation of high-resolution protein localization maps that encapsulate the fundamental characteristics of cells, including elemental cellular localizations such as nuclear and non-nuclear domains. ResultsQualitative and quantitative comparisons were conducted across internal and external clustering evaluation metrics, as well as various facets of the clustering results. The study also explored different components of the model. The research outcomes conclusively indicate that, in comparison to previous methods, Sc2promap exhibits superior performance. ConclusionsThe amalgamation of the attention mechanism and prediction head components has led the model to excel in protein localization clustering and analysis tasks. General significanceThe model effectively enhances the capability to extract features and knowledge from protein fluorescence images.

Probing the Mechanisms Underlying the Transport of the Vinca Alkaloids by P-glycoprotein

The efficacy of many cancer drugs is hindered by P-glycoprotein (Pgp), a cellular pump that removes drugs from cells. To improve chemotherapy, drugs capable of evading Pgp must be developed. Despite similarities in structure, vinca alkaloids (VAs) show disparate Pgp-mediated efflux ratios. ATPase activity and binding affinity studies show at least two binding sites for the VAs: high- and low-affinity sites that stimulate and inhibit the ATPase activity rate, respectively. The affinity for ATP from the ATPase kinetics curve for vinblastine (VBL) at the high-affinity site was 2- and 9-fold higher than vinorelbine (VRL) and vincristine (VCR), respectively. Conversely, VBL had the highest Km (ATP) for the low-affinity site. The dissociation constants (KDs) determined by protein fluorescence quenching were in the order VBL < VRL< VCR. The order of the KDs was reversed at higher substrate concentrations. Acrylamide quenching of protein fluorescence indicate that the VAs, either at 10 µM or 150 µM, predominantly maintain Pgp in an open-outward conformation. When 3.2 mM AMPPNP was present, 10 µM of either VBL, VRL, or VCR cause Pgp to shift to an open-outward conformation, while 150 µM of the VAs shifted the conformation of Pgp to an intermediate orientation, between opened inward and open-outward. However, the conformational shift induced by saturating AMPPNP and VCR condition was less than either VBL or VRL in the presence of AMPPNP. At 150 µM, atomic force microscopy (AFM) revealed that the VAs shift Pgp population to a predominantly open-inward conformation. Additionally, STDD NMR studies revealed comparable groups in VBL, VRL, and VCR are in contact with the protein during binding. Our results, when coupled with VAs-microtubule structure-activity relationship studies, could lay the foundation for developing next-generation VAs that are effective as anti-tumor agents. A model that illustrates the intricate process of Pgp-mediated transport of the VAs is presented.

Insights on the in-vitro binding characteristics of human α-1-acid glycoprotein (HAG) with JAK inhibitor ocalcitinib: Multi-spectroscopic analysis combined with theoretical calculations

A powerful Janus kinase (JAK) inhibitor, ocalcitinib has been authorized for controlling pruritus and atopic dermatitis from canine allergic dermatitis and many kinds of cytokines. To further understand the drug action, the interaction between ocalcitinib and HAG was explored using multi-spectroscopic approaches combined with theoretical calculations. The results demonstrated that ocalcitinib effectively quenched the intrinsic fluorescence of HAG protein through a mixed quenching and formed a stable ocalcitinib–HAG complex that had the association constant of 104–105M−1, outlining that ocalcitinib binding to HAG protein had a moderate affinity. In the ocalcitinib–HAG complex, ocalcitinib implanted into the bucket-shaped hydrophobic chamber of HAG, leading to the slight variety in the hydrophobicity of the microenvironment encircled Trp-and Tyr-resides as well as the secondary structure of HAG. And, the driving forces that forms the ocalcitinib-HAG complex were mainly the hydrogen bonding interaction, Van der Waals forces, and hydrophobic interaction. It is also confirmed that the common metal ions like Ca2+, Mg2+, Fe3+, Zn2+, Cu2+, Co2+, Ni2+ ions) except K+ ion had a greater negative influence on the complexation between ocalcitinib and HAG. This study should conducive to estimating the pharmacological natures of ocalcitinib, comprehending the distribution and operation of the drug in the body, and developing novel medication devise.

Fluorescence study of the interaction between albumin and layered double hydroxides

Layered double hydroxides nanoparticles (LDH-NP) are increasingly studied for biomedical applications. Nevertheless, their interaction with biomolecules such as proteins needs further exploration for an effective application. In this work, the adsorption of bovine serum albumin (BSA) on LDH-NP and the conformation changes of the protein upon adsorption were characterized using fluorescence spectroscopy. First, the quenching of tryptophan residues of BSA by chloride-intercalated LDH-NP was explored and the BSA adsorption capacity of LDH-NP were determined. Then, the structural conformation of the protein was analyzed by fluorescence spectroscopy (including synchronous, polarization and quenching studies) at different surface coverages. Finally, the proclivity of adsorbed BSA molecules to assemble as amyloid fibril was evaluated. Due to the positive charging and low curvature of LDH-NP, BSA molecules were strongly adsorbed, which produced a quenching of the protein fluorescence and a large adsorption capacity. The effect on BSA conformation was dependent on surface coverage (SC): at low values ,t he tryptophan residues were in more hydrophobic environments and more accessible to quenchers than al high ones. At low SC, there is space between the BSA molecules to spread on the surface, which led to a conformation change. Contrarily, the native conformation around tryptophan residues of BSA was preserved at high SC due to the tight packing of the adsorbed protein molecules. As a result, BSA molecules are stabilized against the formation of amyloid fibrils at high SC, while at low SC they present a similar fibrillation than free BSA.