Molecular Fluorescence Spectroscopy Research Articles

A structural and biochemical approach to effect of temperature and pH on the Molten Globule phenomenon in a thermophilic protease: Molecular dynamics simulation, fluorescence spectroscopy and circular dichroism

Proteins in the molten globule (MG) stage have a reasonably stable secondary structure content, but changes in side-chain packing cause a noticeable decrease in the number of tertiary structures. Here, we investigated serine protease as a suitable model to study the folding route and characteristics of a protein in the MG state. Upon induction with IPTG, the protein exhibited a molecular weight of 42 kDa and achieved a final concentration of 1.7 mg/mL after the concentration process. Serine protease's intrinsic and extrinsic fluorescence was examined in the pH range of 1–12 using fluorescence spectroscopy with maximum intrinsic fluorescence in 335 nm and a blue shift in 510 nm for extrinsic fluorescence. It was suggested that the protein transitions probably to an MG state at pH 3. Evidences showed that serine protease's secondary structure using far-UV circular dichroism (CD) showed a little shift in the MG state intermediate, pH 3, relative to the original state, pH 8, which is typical for MG. Maximum decreases was observed in 228 to 300 nm for UV-near CD. Also revealed a notable alteration in the pH 3 of the enzyme compared to pH 8, which is the pH at which the protein takes on its natural structure. By using acrylamide to quench the fluorescence, the Stern-Volmer constant (KSV) was determined at pH values of 8 and 3, yielding values of 20 and 14 M−1, respectively. This is likely correlated with an increase in the enzyme's molecular volume in the MG state. Molecular dynamics (MD) simulation showed that although the thermal stability of this enzyme at 90° was confirmed in the Root-Mean-Square-Deviation (RMSD), Root-Mean-Square-Fluctuation (RMSF), Radius of gyration (Rg) as well as Solvent-Accessible-Surface-Area (SASA) graphs, but pH 3 may significantly disturb the tertiary structure and secondary structure. These in-silico results may also be analyzed in line with the occurrence of MG at pH 3.

Serotonin Promotes Vesicular Association and Fusion by Modifying Lipid Bilayers.

The primary event in chemical neurotransmission involves the fusion of a membrane-limited vesicle at the plasma membrane and the subsequent release of its chemical neurotransmitter cargo. The cargo itself is not known to have any effect on the fusion event. However, amphiphilic monoamine neurotransmitters (e.g., serotonin and dopamine) are known to strongly interact with lipid bilayers and to affect their mechanical properties, which can in principle impact membrane-mediated processes. Here, we probe whether serotonin can enhance the association and fusion of artificial lipid vesicles in vitro. We employ fluorescence correlation spectroscopy and total internal reflection fluorescence microscopy to measure the attachment and fusion of vesicles whose lipid compositions mimic the major lipid components of synaptic vesicles. We find that the association between vesicles and supported lipid bilayers is strongly enhanced in a serotonin dose-dependent manner, and this drives an increase in the rate of spontaneous fusion. Molecular dynamics simulations and fluorescence spectroscopy data show that serotonin insertion increases the water content of the hydrophobic part of the bilayer. This suggests that the enhanced membrane association is likely driven by an energetically favorable drying transition. Other monoamines, such as dopamine and norepinephrine, but not other related species, such as tryptophan, show similar effects on membrane association. Our results reveal a lipid bilayer-mediated mechanism by which monoamines can themselves modulate vesicle fusion, potentially adding to the control toolbox for the tightly regulated process of neurotransmission in vivo.

Myricetin and dihydromyricetin as JNK1 inhibitors protect against ROS‐mediated oxidative stress and apoptosis

AbstractReactive oxygen species (ROS)‐mediated oxidative stress and apoptosis contribute to the pathogenesis and pathological conditions of various diseases induced by food and environmental pollutants. Myricetin and dihydromyricetin, as natural flavonoids, can counteract oxidative damage, apoptosis, and associated diseases. Molecular docking analysis, ELISA, fluorescence spectroscopy, and Western blot techniques were applied to examine the potential of the two flavonoids as effective therapeutics for ROS‐mediated diseases and the mechanism(s) underlying their protective effects against ethanol/H2O2‐induced damage. In Kunming mice and HepG2 cells, myricetin and dihydromyricetin inhibited ethanol/H2O2‐induced liver damage, ROS accumulation, and oxidative stress and apoptosis. Molecular docking showed that myricetin and dihydromyricetin interacted with JNK1 via hydrophobic interactions and hydrogen bonds, thereby exerting their protective effects. The significant differences in their binding patterns to JNK1 resulted from the different bond types between C2 and C3 on their C ring. Myricetin and dihydromyricetin likely protected liver injury via counteracting ROS/JNK1‐mediated oxidative stress and apoptosis. The finding that the suppression of ROS‐mediated oxidative stress and apoptosis by flavonoids via interacting with functional proteins, such as kinase, may represent an alternative strategy for treating excessive ROS‐induced health problems.

In silico and in vitro Studies Evaluating the Promising Antiureolytic Activity of Schiff’s Base 4-(3-Hydroxybenzylideneamino)phenol and Its Amine Derivative

Urea is the most widely used nitrogen fertilizer worldwide. However, ammonia volatilization, resulting from applying urea to the soil surface, causes economic and environmental losses; thus, urease inhibitors have been developed to mitigate these losses. In this work, the anti-ureolytic activity of Schiff’s base 4-(3-hydroxybenzylideneamino) phenol (3B4) and its amine-derived (3B4a) was evaluated. The most promising urease inhibitor in soil was 3B4 (55.0 ± 3.9% inhibition), with comparable results to N-(butyl) thiophosphoric triamide (p = 0.659). In the in vitro analysis (Canavalia ensiformis), the results of anti-ureolytic activity were similar, 22.6 ± 6.9% for 3B4 and 24.2 ± 9.6% for 3B4a. Biophysical interaction studies were also carried out through molecular docking studies and molecular fluorescence spectroscopy. These studies showed that both substances are preferentially competitive inhibitors, with the interaction between 3B4a and urease forming a more stable complex. In the analysis by Fourier transform infrared spectroscopy, no interaction was observed when 3B4 or 3B4a was mixed with urea (1:1) for 48 h, providing evidence of compatibility. Thus, the Schiff base 3B4 and its corresponding amine 3B4a may represent potential additives for urea fertilization aiming to assist in the urease inhibition process.

Multi-analytical techniques to study changes in carbon and nitrogen forms in a tomato-cultivated soil treated with biochar and biostimulants

Agro-environmental applications of biochar and biochar in combination with biostimulants require a full understanding of the mobility and fate of the carbon and nitrogen fractions in soils. The effects of biochar and biostimulants on forms of nitrogen and carbon in soil during a field-scale incubation were investigated by a multi-analytical approach. This study was conducted on a tomato-cultivated agricultural land treated with low doses of biochar (about 0.1%) and different biostimulants: Micosat F®, arbuscular mycorrhiza fungi (AMF), or a consortium of Pseudomonas fluorescens, Bacillus sp., and a nitrogen-fixing bacteria (Consortium B). Forms of carbon and nitrogen and their mobility before, during, and after tomato growth, were studied with different techniques including elemental analysis, adsorption and molecular fluorescence spectroscopy, ion chromatography, and a column leaching test. Due to the low load of biochar and the short study time, elemental analyses might not be sensitive enough to determine C and N variation in the soil. Based on the dissolved organic carbon (DOC) and dissolved nitrogen forms, the treatments with biochar and biostimulants affected the mobility of these elements with an overall decrease at the end of tomato growth. The organic carbon is mainly ascribable to humic and fulvic acids, as indicated by spectroscopic analysis. The leaching column test demonstrated that cumulative leached C is about one order of magnitude lower than the DOC. In addition, simulated rain cycles profoundly affected their leaching, so it is important to design leaching tests based on local and seasonal weather conditions. In short, positive effects were observed in the marketable production of tomato when soil was treated with biochar combined with a mixture of biostimulants.

Preparation and Performance Characterization of an Active Luminous Coating for Asphalt Pavement Marking

For improving the night recognition of road markings and enhancing the driving safety of asphalt pavements, single-factor optimization is used to investigate the effects of the component materials, including luminescent power, pigment, filler, and anti-sedimentation agent, on the luminous performance of a coating. Additionally, their composition ratios are optimized using response surface methodology. A phosphorescent marking coating is prepared to investigate the micromorphology, excitation, and emission properties using scanning electron microscopy (SEM) and molecular fluorescence spectroscopy (MFS). The optimum thickness of the coating on an asphalt pavement is investigated, and the durability of the coating on asphalt pavement using a wheel rutting test is evaluated. The results show that the 300 mesh yellow-green luminous powder has the optimal overall performance, with an initial luminescence that exceeds that of orange and sky blue by three times. Initial brightness is mainly influenced by aluminate luminescent powder (ALP), which increases with the dosage. ALP and fumed silica powder (FSP) have a positive effect on brightness after centrifugation, and the effect of FSP dosage is more significant. ALP, rutile titanium dioxide powder (RTDP), and FSP influence the wear value of the coating, and the magnitude of the effect is RTDP > FSP > ALP. The optimal dosages of the main component are 27% ALP, 5% RTDP, and 0.8% FSP. The results of SEM show that the components in the coating are evenly dispersed, and the surface of the coating is rough. The peak excitation wavelength of 420 nm means that the coating has the best excitation effect in UV light, and its emission spectrum in the 440–760 nm wavelength range is well within the sensitive recognition zone of the human eye. The initial brightness gradually reached 4.38 cd/m2 when the coating thickness was increased from 482 μm to 546 μm, and the optimal application thickness of the luminous coating was determined to be 500 μm. At high and normal temperatures, the rutting stripping rates of the luminous marking coating are 16.8% and 8.2%, indicating its satisfactory durability. This study provides an experimental basis for the ratio optimization design of a luminous coating for asphalt pavements.

Nozzle Electrode Point Discharge-Enhanced Oxidation and Portable Gas Phase Molecular Fluorescence Spectrometry for Sensitive Field Detection of Sulfide

It is still a challenge to accurately determine dissolved sulfide due to its susceptibility to contamination and loss during transportation, storage, and analysis in the laboratory, thus highlighting the necessity for its sensitive field analysis. Herein, a robust nozzle electrode point discharge (NEPD) enhanced oxidation coupling with chemical vapor generation (CVG) is described for the highly efficient and flameless conversion of sulfide (S2-) to SO2. Subsequently, a portable and low-power consumption gas phase molecular fluorescence spectrometry (GP-MFS) was constructed for the highly selective and sensitive determination of the generated SO2 via detecting its molecular fluorescence excited by a zinc hollow cathode lamp. Under optimal conditions, a limit of detection (LOD) of 0.1 μM was obtained for dissolved sulfide with a relative standard deviation (RSD, n = 11) of 2.6%. The accuracy and practicability of the proposed method were validated by the analyses of two certified reference materials (CRMs) and several river and lake water samples with satisfactory recoveries of 99%-107%. This work confirms that NEPD enhanced oxidation is a low energy consumption yet highly efficient method for the flameless oxidation of hydrogen sulfide and thus is suitable for the easy field detection of dissolved sulfide in environmental water by CVG-GP-MFS.

Detection of pyrimidine-rich DNA sequences based on the formation of parallel and antiparallel triplex DNA and fluorescent silver nanoclusters

In this work, the use of DNA-stabilized fluorescent silver nanoclusters for the detection of target pyrimidine-rich DNA sequences by formation of parallel and antiparallel triplex structures is studied by molecular fluorescence spectroscopy. In the case of parallel triplexes, the probe DNA fragments are Watson-Crick stabilized hairpins, and whereas in the case of antiparallel triplexes, the probe fragments are reverse-Hoogsteen clamps. In all cases, the formation of the triplex structures has been assessed by means of polyacrylamide gel electrophoresis, circular dichroism, and molecular fluorescence spectroscopies, as well as multivariate data analysis methods. The results have shown that it is possible the detection of pyrimidine-rich sequences with an acceptable selectivity by using the approach based on the formation of antiparallel triplex structures.

Cyclodextrin-Based Displacement Strategy of Sterigmatocystin from Serum Albumin as a Novel Approach for Acute Poisoning Detoxification

This study demonstrates that sterigmatocystin (STC) interacts non-covalently with various cyclodextrins (CDs), showing the highest binding affinity for sugammadex (a γ-CD derivative) and γ-CD, and an almost order of magnitude lower affinity for β-CD. This difference in affinity was studied using molecular modelling and fluorescence spectroscopy, which demonstrated a better insertion of STC into larger CDs. In parallel, we showed that STC binds to human serum albumin (HSA) (a blood protein known for its role as a transporter of small molecules) with an almost two order of magnitude lower affinity compared to sugammadex and γ-CD. Competitive fluorescence experiments clearly demonstrated an efficient displacement of STC from the STC-HSA complex by cyclodextrins. These results are a proof-of-concept that CDs can be used to complex STC and related mycotoxins. Similarly, as sugammadex extracts neuromuscular relaxants (e.g., rocuronium and vecuronium) from blood and blocks their bioactivity, it could also be used as first aid upon acute intoxication to encapsulate a larger part of the STC mycotoxin from serum albumin.

Effect of Catechin on Yolk Immunoglobulin Structure and Properties: A Polyphenol-Protein Interaction Approach.

The preparation of the interaction between polyphenols and protein is of great significance for increasing added value and promoting the application of egg yolk immunoglobulin (IgY). This study systematically investigated the effect of catechin on yolk immunoglobulin structural characteristics and functional properties. The binding conditions, force types, molecular conformation, and residual microenvironment of the interaction between catechin and IgY were analyzed by molecular docking technology, UV-vis absorption and fluorescence spectroscopy studies. The results showed that the main binding forces in the complex were hydrogen bonding and van der Waals forces. After the interaction, fluorescence quenching occurred and the maximum emission wavelength was redshifted. The results showed that the microenvironment around IgY increased polarity, increased hydrophilicity and decreased hydrophobicity, and the structure of the peptide chain changed. The bacteriostatic thermal stability of the compound against Escherichia coli and Staphylococcus aureus was lower than that of catechin IgY. The bacteriostatic acid and base stability were higher than that of catechin and IgY. The antioxidant activity was catechin, complex, and IgY, in descending order. The antioxidant activity of catechin and complex was significantly higher than that of IgY. At the same concentration, the apparent viscosity of the three samples was complex, IgY and catechin, in descending order. G' was greater than G" indicating that elastic properties dominate in G". The G' and G" values of the complex were higher than those of the other groups. Rheological results indicated that the complex may have high physical stability. This study provides theoretical support for broadening the application field of IgY and suggest its properties change in the machining process. It also provides new ideas for the development of functional foods from poultry eggs.

Spectrometric study of the interaction of phycocyanins with egg proteins

Phycocyanins isolated from the biomass of Spirulina platensis are biologically active substances with antioxidant, anti-inflammatory and immunomodulatory properties. At the Federal State Budgetary Institution “Federal Research Center for Nutrition and Biotechnology” research is being conducted to create a food module - a carrier of these biologically active substances. In this work, the possible formation of contacts of phycocyanins with egg proteins was studied using molecular fluorescence spectroscopy. As a result of the studies, it was shown that a mixture of egg white and phycocyanin has a fluorescence maximum in the region characteristic of egg white (361 nm; 125 a.u.) and the fluorescence of phycocyanin has not been established. The results of the study indicate the formation of a bond between phycocyanin and the amino acids that make up the egg white, followed by shielding of phycocyanin molecules from the action of exciting radiation.

Alzheimer's disease diagnosis by blood plasma molecular fluorescence spectroscopy (EEM)

Despite tremendous research advances in detecting Alzheimer's disease (AD), traditional diagnostic tests remain expensive, time-consuming or invasive. The search for a low-cost, rapid, and minimally invasive test has marked a new era of research and technological developments toward establishing blood-based AD biomarkers. The current study has employed excitation-emission matrices (EEM) of fluorescence spectroscopy combined with machine learning to diagnose AD using blood plasma samples from 230 individuals (83 AD patients from 147 healthy controls). To evaluate the performance of the classification algorithms, we calculated the commonly used figures of merit (accuracy, sensitivity and specificity) and figures of merit that take into account the samples unbalance and the discrimination power of the models, as F2-score (F2), Matthews correlation coefficient (MCC) and test effectiveness (delta). The classification models achieved satisfactory results: Parallel Factor Analysis with Quadratic Discriminant Analysis (PARAFAC-QDA) with 83.33% sensitivity, 100% specificity, 86.21% F2; and Tucker3-QDA with 91.67% sensitivity, 95.45% specificity and 91.67% F2. In addition, the classifiers show high overall performance with 94.12% accuracy and 0.87 MCC. Regarding the discrimination power between healthy and AD patients, the classification algorithms showed high effectiveness with the mean scores separated by three or more standard deviations. The PARAFAC's spectral profiles and the wavelength values from both models loading profiles can be used in future research to relate this information to plasma AD biomarkers. Our results point to a rapid, low-cost and minimally invasive blood-based method for AD diagnosis.

The dynamic interplay of PIP2 and ATP in the regulation of the KATP channel.

ATP-sensitive potassium (KATP ) channels couple the intracellular ATP concentration to insulin secretion. KATP channel activity is inhibited by ATP binding to the Kir6.2 tetramer and activated by phosphatidylinositol 4,5-bisphosphate (PIP2 ). Here, we use molecular dynamics simulation, electrophysiology and fluorescence spectroscopy to show that ATP and PIP2 occupy different binding pockets that share a single amino acid residue, K39. When both ligands are present, simulations suggest that K39 shows a greater preference to co-ordinate with PIP2 than with ATP. They also predict that a neonatal diabetes mutation at K39 (K39R) increases the number of hydrogen bonds formed between K39 and PIP2 , potentially accounting for the reduced ATP inhibition observed in electrophysiological experiments. Our work suggests that PIP2 and ATP interact allosterically to regulate KATP channel activity. KEY POINTS: The KATP channel is activated by the binding of phosphatidylinositol 4,5-bisphosphate (PIP2 ) lipids and inactivated by the binding of ATP. K39 has the potential to bind to both PIP2 and ATP. A mutation to this residue (K39R) results in neonatal diabetes. This study uses patch-clamp fluorometry, electrophysiology and molecular dynamics simulation. We show that PIP2 competes with ATP for K39, and this reduces channel inhibition by ATP. We show that K39R increases channel affinity to PIP2 by increasing the number of hydrogen bonds with PIP2 , when compared with the wild-type K39. This therefore decreases KATP channel inhibition by ATP.

Green hemi-synthesis of novel thiazole derivatives from Ammodaucus leucotrichus Coss. & Dur. and Cuminum cyminum L. essential oils: stereochemistry, molecular fluorescence spectroscopy, in vitro biologicial activity, and molecular docking study

This paper reports the hemi-synthesis of a new thiazole derivatives from 3-allylrhodanine under green chemical conditions (solvent free and ambient temperature). Two targeted molecules: perillaldehyde and cuminaldehyde from essential oils of Ammodaucus leucotrichus Coss. & Dur. and Cuminum cyminum L. were used. Thiazole derivatives (HRP11, 12) were obtained and characterized by FTIR, 1H NMR, 13C NMR, 2D NMR. In addition, stereochemistry of HRP11, 12 was characterized by using 2D NMR and their structural stability was confirmed by DFT calculation. Furthermore, the new products were screened for their in vitro biological activity and in silico interaction with TNF-α by using molecular docking study. A moderate antioxidant activity was found for the two obtained products evaluated by DPPH and ABTS radical scavenging techniques. Against all odds, HRP12 revealed an interesting antibacterial activity against Escherichia coli, Citrobacter freundii, and Salmonella typhimurium. Thereby, a molecular fluorescence spectroscopy was carried out to compare the fluorescence of gentamicin and that of HRP11, 12. Molecular docking performed on the Tumor Necrosis Factor (TNF-α) demonstrated sensitivity towards the thiazole derivatives obtained.

Evidence of humic acid-aluminium‑silicon complexes under controlled conditions

The chemistry of silicon (Si), the second most abundant element in soil after oxygen, is not yet fully understood in the soil-water-plant continuum. Although Si is widely accepted as an element that has little or no interaction with natural organic matter, some data seems to show the opposite. To identify a potential interaction between natural organic matter and Si, batch experiments were achieved at various pH and Si concentrations, involving also Al3+ as a common ion in soil and using humic acid (HA) as a typical model for natural organic matter. Several complementary techniques were used to characterize the possible complexes formed in the dissolved or solid phases: molecular fluorescence spectroscopy, 29Si solid-state NMR, Fourier transform infrared spectroscopy, quantification of Si, Al and organic carbon, and nanoparticle size distribution. These tools revealed that humic acid indeed interacts, but weakly, with Si alone. In the presence of Al, however, a ternary complex HA-Al-Si forms, likely with Al as the bridging atom. The presence of Si promotes the maintenance of both Al and dissolved organic matter (DOM) in solution, which is likely to modify the result or the kinetics of pedogenesis. Such complexes can also play a role in the control of Al toxicity towards plants and probably also exists with other metals, such as Fe or Mn, and other metalloids such as As.

Interactions between Rhodamine Dyes and Model Membrane Systems-Insights from Molecular Dynamics Simulations.

Background: rhodamines are dyes widely used as fluorescent tags in cell imaging, probing of mitochondrial membrane potential, and as P-glycoprotein model substrates. In all these applications, detailed understanding of the interaction between rhodamines and biomembranes is fundamental. Methods: we combined atomistic molecular dynamics (MD) simulations and fluorescence spectroscopy to characterize the interaction between rhodamines 123 and B (Rh123 and RhB, respectively) and POPC bilayers. Results: while the xanthene moiety orients roughly parallel to the membrane plane in unrestrained MD simulations, variations on the relative position of the benzoic ring (below the xanthene for Rh123, above it for RhB) were observed, and related to the structure of the two dyes and their interactions with water and lipids. Subtle distinctions were found among different ionization forms of the probes. Experimentally, RhB displayed a lipid/water partition coefficient more than two orders of magnitude higher than Rh123, in agreement with free energy profiles obtained from umbrella sampling MD. Conclusions: this work provided detailed insights on the similarities and differences in the behavior of bilayer-inserted Rh123 and RhB, related to the structure of the probes. The much higher affinity of RhB for the membranes increases the local concentration and explains its higher apparent affinity for P-glycoprotein reconstituted in model membranes.

Oxidation of small aromatic compounds in rainwater by UV/H2O2: Optimization by response surface methodology

The presence of aromatic compounds in rainwater is a matter of concern, mainly when the use of rainwater in buildings is intended. The present work aimed to assess the oxidation of a mixture of small aromatic compounds (benzoic, 3,5-dihydroxybenzoic and syringic acids) in rainwater by the UV/H2O2 process, and the possibility of its optimization by the response surface methodology. The extent of oxidation was assessed by ultraviolet-visible and molecular fluorescence spectroscopies. During the oxidation of the mixture new chromophoric compounds were formed at an initial stage, but they were then degraded at a later stage. The increase of the H2O2 concentration, resulted in a higher extent of oxidation, while the initial pH value showed no influence in the oxidation of the mixture. The optimization of the oxidation was performed using the uniform design with the factors: initial H2O2 concentration, initial pH, and reaction time. The response surface model found, through the best subsets regression, described the extent of oxidation as function of the following variables: initial H2O2 concentration and reaction time, interaction between them, and also their respective quadratic forms. The optimal conditions, the lowest H2O2 concentration (3.1 mM) for a selected maximum reaction time (4 h), were applied to rainwater samples spiked with the mixture of contaminants and resulted in an extent of oxidation higher than 99.5%, validating the application of the model to real samples. Therefore, the UV/H2O2 process coupled to its optimization via response surface methodology may be an alternative for rainwater treatment in buildings.

Structure and stability of 7-mercapto-4-methylcoumarin self-assembled monolayers on gold: an experimental and computational analysis.

Self-assembled monolayers (SAM) of 7-mercapto-4-methylcoumarin (MMC) on a flat gold surface were studied by molecular dynamics (MD) simulations, reference-free grazing incidence X-ray fluorescence (GIXRF) and X-ray photoelectron spectroscopy (XPS), to determine the maximum monolayer density and to investigate the nature of the molecule/surface interface. In particular, the protonation state of the sulfur atom upon adsorption was analyzed, since some recent literature presented evidence for physisorbed thiols (preserving the S-H bond), unlike the common picture of chemisorbed thiyls (losing the hydrogen). MD with a specifically tailored force field was used to simulate either thiol or thiyl monolayers with increasing number of molecules, to determine the maximum dynamically stable densities. This result was refined by computing the monolayer chemical potential as a function of the density with the bennet acceptance ratio method, based again on MD simulations. The monolayer density was also measured with GIXRF, which provided the absolute quantification of the number of sulfur atoms in a dense self-assembled monolayer (SAM) on flat gold surfaces. The sulfur core level binding energies in the same monolayers were measured by XPS, fitting the recorded spectra with the binding energies proposed in the literature for free or adsorbed thiols and thiyls, to get insight on the nature of the molecular species present in the layer. The comparison of theoretical and experimental SAM densities, and the XPS analysis strongly support the picture of a monolayer formed by chemisorbed, dissociated thiyls.

High-resolution MD Simulation Studies to Get Mechanistic Insights into the Urea-induced Denaturation of Human Sphingosine Kinase 1.

Sphingosine kinase 1 (SPhK1) is a crucial signaling enzyme involved in cell proliferation, cellular survival, stimulation of angiogenesis, and apoptosis prevention. Recently, we have reported the unfolding kinetics of SPhK1 using molecular dynamics (MD) simulation, circular dichroism, and fluorescence spectroscopy. We found that SPhK1 showed a biphasic unfolding with an intermediate state (~ 4.0 M urea). We aim to understand the impact of MD simulation duration on the structure, function, and dynamics of proteins. In order to get deeper insights into the folding mechanism, an extended MD simulation is required. Here, we extended the MD simulations time scale from 100 to 300 ns on SPhK1 at increasing urea concentration to explore structural changes in the SPhK1. The results suggested a constant form of the unfolding of SPhK1 upon extending the simulation time scale at different urea concentrations. Furthermore, we showed step by step unfolding and percentage of secondary structure contents in SPhK1 under the influence of urea at each concentration. The results from the current work revealed a uniform pattern of the SPhK1 unfolding at different urea concentrations. This study provides deeper mechanistic insights into the urea-induced denaturation of SPhK1.

Adsorption of arabinogalactan-proteins from Acacia gums (senegal and seyal) and its molecular fractions onto latex particles

Adsorption of Acacia gums (GA) (A. senegal and A. seyal) and A. senegal molecular fractions, made of arabinogalactan-proteins (AGPs) with different molecular weights, onto latex particles, with different sizes and surface charges, was studied using molecular (fluorescence and nanoIR spectroscopies, microscopies including Atomic Force Microscopy) and mesoscopic (Dynamic Light Scattering, zeta potential, Asymmetrical flow field-flow fractionation) methods. Adsorption of GA onto latex particles revealed a complex mechanism of adsorption where the highest adsorption was observed when A. senegal and latex particles were negatively charged. The invert was observed for A. seyal where the maximum of adsorption was observed when gum and latex were oppositely charged. The duality of positive and negative charges on GA macromolecules together with conformational changes certainly explain the complex mechanism of adsorption. A mechanism where either the protein moiety either the sugars blocks adsorb is to be preferred. This mechanism seems to produce differences of behaviour due to viscoelastic properties of adsorbed layers, and differences of hydration due to differences of polarity of AGPs. The consequence of electrostatics in the adsorption process is a water release from the adsorbed layers and important gain in entropy. A fair agreement was found between protein content and adsorbed layer thickness of GA, with the formation of partial coverage, monolayer and multilayer films depending on GA concentration. Structural changes induced by GA adsorption onto latex particles was probed using fluorescence and nanoIR spectroscopies without giving clear evidence of conformational changes induced after GA adsorption. The results of this study highlighted that GA surface adsorption process depend not only on the protein moiety and electrostatic interactions but also on other parameters related to AGP hydration status. The protein structural accessibility, the molecular weight distribution, the AGPs intrinsic viscoelastic properties allowing structural rearrangements on the surface and spreading in order to form a viscoelastic film onto latex particles should also play a pivotal role in the adsorption process.