Binding of flavonoids to yeast aldehyde dehydrogenase: a molecular mechanism and computational approach.

The interaction of three flavonoids, apigenin, fisetin and quercetin with yeast aldehyde dehydrogenase, ALDH was studied by spectroscopic and molecular docking methods. A combination of both static and dynamic processes interaction mechanism for the binding of flavonoids with ALDH was found. The interaction takes place with moderate binding and the interaction was driven by hydrophobic contacts. The microenvironments of the fluorescent amino acids changed upon flavonoids binding. The distances between ALDH and flavonoids determined by Förster Resonant Energy Transfer (FRET) confirmed the results obtained by fluorescence. The structure of ALDH against thermal denaturation was stabilized by apigenin and destabilized by fisetin and quercetin. Molecular docking simulation showed that all flavonoids bind to the same site of ALDH and confirmed the moderate binding straight found in fluorescence.Communicated by Ramaswamy H. Sarma.

Modulation of the structural and functional properties of α1-antitrypsin by interaction with flavonoid luteolin.

α1-antitrypsin (A1AT) is a circulating serine protease inhibitor and an acute phase reactant, the deficiency of which can lead to liver failure and chronic lung disease. Flavonoid treatment may induce changes in α1-antitrypsin production in some human cells. The purpose of this study is to investigate the properties of the A1AT protein that interacts with the flavonoid luteolin, which exhibits numerous properties, including antioxidant properties. For this purpose, multi-spectroscopic (UV-Vis spectroscopy, fluorescence and FRET) methods and molecular docking were used. The intrinsic fluorescence of A1AT was quenched by luteolin through a static mechanism. Luteolin binds to one site of the A1AT protein, with a moderate binding constant, and the binding process was driven by entropy and hydrophobic interactions. Hydrophobicity around Trp decreased as a result of luteolin binding to the A1AT site and FRET occurred at a distance of 3.11 nm. Under the action of temperature, the stability of A1AT structure was decreased by the presence of luteolin. Molecular docking confirmed that luteolin binds to one site, with a moderate affinity. The results would give a better understanding of the functional changes that occurred in the structure of A1AT induced by luteolin binding, which may have implications in the field of pharmaceutical research.Communicated by Ramaswamy H. Sarma.

Conformational Changes in the BSA-LT4 Complex Induced by the Presence of Vitamins: Spectroscopic Approach and Molecular Docking.

Levothyroxine (LT4) is known for its use in various conditions including hypothyroidism. LT4 interaction with serum albumin may be influenced by the presence of vitamins. For this reason, we investigated the effect of vitamin C, vitamin B12, and folic acid on the complex of Bovine Serum Albumin with LT4 (BSA-LT4). UV-Vis spectroscopy was used to monitor the influence of vitamins on the BSA-LT4 complex. Fluorescence spectroscopy revealed a static quenching mechanism of the fluorescence of BSA-LT4 complex by the vitamin C and folic acid and a combined mechanism for vitamin B12. The interaction of vitamin C and folic acid with BSA-LT4 was moderate, while the binding of vitamin B12 was much stronger, extending the storage time of LT4 in blood plasma. Synchronous fluorescence found that the vitamins were closer to the vicinity of Trp than to Tyr and the effect was more pronounced for the binding of vitamin B12. The thermal stability of the BSA-LT4 complex was more evident, but no influence on the stability of BSA-LT4 complex was obtained for vitamin C. Molecular docking studies showed that vitamin C and folic acid bound the same site of the protein, while vitamin B12 bonded to a different site.

Biogenic Ferrihydrite Nanoparticles Produced by Klebsiella oxytoca: Characterization, Physicochemical Properties and Bovine Serum Albumin Interactions.

The synthesis of nanoparticles inside microorganisms is an economical alternative to chemical and physical methods of nanoparticle synthesis. In this study, ferrihydrite nanoparticles synthesized by Klebsiella oxytoca bacterium in special conditions were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDS), small-angle X-ray (SAXS), UV-Vis spectroscopy, fluorescence, fluorescence resonance energy transfer (FRET), and molecular docking. The morphology and the structure of the particles were characterized by means of SEM and SAXS. The elemental content was determined by means of the EDS method. The absorption properties of the ferrihydrite nanoparticles were investigated by UV-Vis spectroscopy. The binding mechanism of the biogenic ferrihydrite nanoparticles to Bovine Serum Albumin (BSA) protein, studied by fluorescence, showed a static and weak process, combined with FRET. Protein denaturation by temperature and urea in the presence of the ferrihydrite nanoparticles demonstrated their influence on the unfolding process. The AutoDock Vina and UCSF Chimera programs were used to predict the optimal binding site of the ferrihydrite to BSA and to find the location of the hydrophobic cavities in the sub-domain IIA of the BSA structure.

Evaluation of the interaction of levothyroxine with bovine serum albumin using spectroscopic and molecular docking studies.

Bovine serum albumin (BSA) acts as a carrier for many endogenous and exogenous compounds, such as thyroid hormones or corresponding drugs. Binding of the hydrophilic levothyroxine drug (LT4) to BSA is of significant pharmacological importance. In this work, UV-vis measurements were used to determine the pH value at which LT4 interacts optimally with proteins. The binding mechanism and affinity of the interaction between LT4 and BSA were investigated using Fourier-transform infrared spectroscopy (FT-IR), fluorescence, fluorescence resonance energy transfer (FRET), Surface Plasmon Resonance (SPR), supplemented by molecular docking analysis. Fluorescence measurements revealed the quenching effect of LT4 on the BSA intrinsic fluorescence and LT4 binding with BSA is driven by a ground-state complex formation that may be accompanied by a nonradiative energy transfer process. The thermodynamic parameters correspond to an enthalpic process, driven mainly by hydrogen bonds and van der Waals forces. Using SPR, the adsorbed amount of biomolecules was calculated and the binding affinity of LT4 with confined-BSA was characterized, indicating that the BSA immobilization plays an important role in LT4 binding. Docking studies confirmed the formation of the LT4-BSA complex with LT4 bound to site I on the BSA structure mainly with amino acid residues Trp 213, Tyr 137, Tyr 147. The calculation of the apparent association constant confirms the result obtained in SPR.Communicated by Ramaswamy H. Sarma.

Probing the interaction of fisetin with human serum transferrin via spectroscopic and molecular docking approaches.

The binding of fisetin to human serum transferrin (HST) was investigated by spectroscopic (steady-state fluorescence, synchronous fluorescence, Förster resonance energy transfer) and molecular docking approaches. HST fluorescence is quenched by fisetin by a static process. The binding takes place with a moderate affinity and it is driven by hydrogen bonding and van der Waals forces. Synchronous fluorescence study indicates that Trp is more involved in the fluorescent quenching of HST by fisetin than Tyr. The energy transfer between HST and fisetin occurs at a distance of 2.31 nm confirming the results obtained by fluorescence. The binding of fisetin to HST favors thermal denaturation of HST conformation. The transition temperature for HST was obtained at 53.81 °C while the presence of the fisetin led to its change to 49.06 °C. The molecular docking of fisetin to HST confirms the results obtained by the spectroscopic experiments showing a moderate affinity of fisetin for HST.Communicated by Ramaswamy H. Sarma.

Molecular insights into binding mechanism of rutin to bovine serum albumin - Levothyroxine complex: Spectroscopic and molecular docking approaches.

Bovine serum albumin (BSA) has been used as a transporter protein for levothyroxine (LT4) and rutin, due to its property of binding to various ligands. Rutin binding to the BSA-LT4 complex can bring many benefits due to its proven pharmacological properties. Using Fourier-Transform Infrared Spectroscopy (FT-IR) the changes induced by rutin in the structure of BSA-LT4 complex were determined. Fluorescence studies allowed us to determine the quenching mechanism and affinity of rutin to the BSA-LT4 complex. The thermodynamic parameters suggest the binding of rutin to BSA-LT4 is a spontaneous process, driven by enthalpy and electrostatic forces. Also, the second derivative of the emission spectra suggests the Trp's of BSA are located in two different microenvironments. Thermal and chemical denaturation of BSA-LT4-rutin complex presents similar behavior but with better stability of the complex in case of chemical denaturation. Molecular docking studies show the binding of the two ligands to the same BSA site, suggesting that rutin may influence the bond of LT4 with the protein. Studies on the antioxidant activity of the BSA-LT4-rutin complex suggest that the presence of LT4 decreases the antioxidant activity of the rutin, but even so this antioxidant activity can be used to bring benefits for medical purposes.

Interactions of Chemically Synthesized Ferrihydrite Nanoparticles with Human Serum Transferrin: Insights from Fluorescence Spectroscopic Studies.

Human serum transferrin (HST) is a glycoprotein involved in iron transport that may be a candidate for functionalized nanoparticles to bind and target cancer cells. In this study, the effects of the simple and doped with cobalt (Co) and copper (Cu) ferrihydrite nanoparticles (Fh-NPs, Cu-Fh-NPs, and Co-Fh-NPs) were studied by spectroscopic and molecular approaches. Fluorescence spectroscopy revealed a static quenching mechanism for all three types of Fh-NPs. All Fh-NPs interacted with HST with low affinity, and the binding was driven by hydrogen bonding and van der Waals forces for simple Fh-NPs and by hydrophobic interactions for Cu-Fh-NPs and Co-Fh-NPs binding, respectively. Of all samples, simple Fh-NPs bound the most to the HST binding site. Fluorescence resonance energy transfer (FRET) allowed the efficient determination of the energy transfer between HST and NPs and the distance at which the transfer takes place and confirmed the mechanism of quenching. The denaturation of the HST is an endothermic process, both in the case of apo HST and HST in the presence of the three types of Fh-NPs. Molecular docking studies revealed that Fh binds with a low affinity to HST (Ka = 9.17 × 103 M-1) in accord with the fluorescence results, where the interaction between simple Fh-NPs and HST was described by a binding constant of 9.54 × 103 M-1.

Structural and molecular aspects of flavonoids as ligands for serum transferrin.

Human serum transferrin (HST) acts as a carrier for Fe3+ and other ions. Binding of flavonoids to HST produces changes in the protein structure with direct implication on iron delivery into cells. We investigate the binding mechanism and affinity towards HST of three flavonoids: rutin, luteolin, and apigenin by different techniques: UV-Vis, fluorescence, fluorescence resonance energy transfer (FRET) combined with molecular docking. UV-Vis results indicate an interaction between flavonoids and HST. It was observed that HST fluorescence was quenched by these three flavonoids via a static process. All the interactions were moderate and the main driving forces are hydrophobic (ΔH > 0 and ΔS > 0) for rutin and luteolin binding or electrostatic (ΔH < 0 and ΔS > 0) for apigenin binding. FRET and molecular docking studies confirm the fluorescence static quenching mechanism by flavonoid binding. The binding of all three flavonoids increases HST stability. These results present the potential use of HST in target-oriented delivery of flavonoids and possibly other drugs into cells.

Ferrihydrite nanoparticles insights: Structural characterization, lactate dehydrogenase binding and virtual screening assay.

The binding between the enzyme lactate dehydrogenase (LDH) and ferrihydrite nanoparticles (Fh-NPs) was investigated by means of small-angle neutron scattering (SANS), Fourier-transform infrared (FTIR) spectroscopy, fluorescence and Förster resonance energy transfer (FRET) and molecular docking. Fh-NPs - LDH compounds of dimensions under 100 nm are formed. The conformational changes and the mechanism of interaction between LDH and Fh-NPs simple and doped with Cu and Co, and the effect of these NPs on the thermal denaturation of LDH were monitored. The quenching mechanism is static, the binding occurring with moderate affinity, being mainly driven by hydrogen bonding and van der Waals forces. FRET occurs at a minimal distance of 2.55 nm. Thermal denaturation of LDH in the presence of simple and doped Fh-NPs shows that the thermodynamic parameters of protein unfolding are significantly changed with temperature. The denaturation temperature of LDH shifts to higher values in the presence of all Fh-NPs, than in the case of simple LDH. The docking approach estimates the energy corresponding to the best fit of the ferrihydrite in the LDH binding site near Trp. These results have direct implications on the uses of the complex of LDH with Fh-NPs in various biochemical, biological, or clinical applications.

Monitoring biomolecular interaction between folic acid and bovine serum albumin.

Folic acid is a bioactive food component whose deficiency can lead to a variety of health problems, while a high intake of folic acid can reduce the cytotoxicity of natural killer cells. The binding mechanism of folic acid to free bovine serum albumin (BSA) was studied using fluorescence, while the biomolecular interaction between confined-BSA and free folic acid was assessed by electrochemical methods and surface plasmon resonance. The fluorescence quenching mechanism of BSA by folic acid was found to have a static character. The thermodynamic parameters of the interaction were determined and indicated a spontaneous exothermic process with a binding constant of 8.72 × 104 M-1 at 25 °C. Confinement of BSA to gold surfaces occurred through different immobilization methods (static and hydrodynamic), inducing conformational changes, which influenced the orientation of BSA molecules binding sites towards free folic acid. The apparent binding constant using electrochemical methods (voltammetry and impedance spectroscopy) was only 5 times higher (41 and 37 × 104 M-1) compared to BSA free in solution, while for surface plasmon resonance, where the hydrodynamic immobilization method was used, the value was much higher (19 × 106 M-1). This work gives also an insight on the interaction of BSA with gold substrates, surface plasmon resonance enabling the calculation of the adsorbed amount. The obtained results help understanding the specific interaction between free and confined BSA with free folic acid.

Exploring the Conformation and Thermal Stability of Human Serum Albumin Corona of Ferrihydrite Nanoparticles.

In the last few years, a great amount of attention has been given to nanoparticles research due to their physicochemical properties that allow their use in analytical instruments or in promising imaging applications on biological systems. The use of ferrihydrite nanoparticles (Fh-NPs) in practical applications implies a particular control of their magnetic properties, stability, biocompatibility, interaction with the surface of the target, and low toxicity. In this study, the formation and organization of human serum albumin (HSA) molecules around the simple Fh-NPs and Fh-NPs doped with Co and Cu were examined by Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) in terms of morphology and particle size. The topology of all Fh-NPs shows an organized area of HSA around each type of Fh-NP. Molecular docking studies were used in order to determine the probable location of the ferrihydrite in the HSA structure. The thermal stability of these nanohybrids was further investigated by fluorimetry, using 214-Trp residue from HSA as a spectral sensor. The denaturation temperature (Tm) was determined, and stabilization of the HSA structure in the presence of Fh-NPs was discussed. This study could be a starting point for the development of different applications targeting the structure and stability of Fh-NPs complexes with proteins.

Ferrihydrite nanoparticles interaction with model lipid membranes.

In recent years was observed an increased interest towards the use of metal nanoparticles for various biomedical applications, such as therapeutics, delivery systems or imaging. As biological membranes are the first structures with which the nanoparticles interact, it is necessary to understand better the mechanisms governing these interactions. In the present paper we aim to characterize the effect of three different ferrihydrite nanoparticles (simple or doped with cooper or cobalt) on the fluidity of model lipid membranes. First we evaluated the physicochemical properties of the nanoparticles: size and composition. Secondly, their effect on lipid membranes was also evaluated using Laurdan, TMA-DPH and DPH fluorescence. Our results can help better understand the mechanisms involved in nanoparticles and membrane interactions.

Insight into the interaction of human serum albumin with folic acid: A biophysical study.

Folic acid (FA) is a water soluble bioactive food constituent from the vitamin B-family complex (B9). FA deficiency can lead to a variety of human health problems, while a high intake of FA can reduce the cytotoxicity of natural killer cells. The main goal of this study was to investigate the interaction of FA with human serum albumin (HSA) at physiological pH using ATR-FTIR, fluorescence spectroscopy, cyclic voltammetry and electrochemical impedance spectroscopy in order to understand the role of HSA as a blood transporter for FA in aqueous solution that can be used in different therapies. The quenching of HSA in the presence of FA was followed and the binding constant (Kb) was determined. The variation of electrochemical parameters proved that the FA binds to immobilized HSA and the binding constant was ten times than the value obtained when the interaction takes place between free molecules in solution when studied by fluorescence quenching. The results can be used in future studies to improve drug delivery systems or cellular uptake of folic acid and food components conjugated to HSA nanoparticles or nanocapsules.