Search

This study investigates the interaction between daphnetin and ovalbumin (OVA) as well as its potential to inhibit OVA fibrillation using both spectroscopic and computational analysis. A moderate binding affinity of 1 × 104M–1was observed between OVA and daphnetin, with a static quenched mechanism identified during the fluorescence quenching processes. Metal ions’ (Cu2+and Zn2+) presence led to an increase in the binding affinities of daphnetin toward OVA, mirroring a similar trend observed with the pH variation. Synchronous and 3D fluorescence studies indicated an increase in the polarity of the microenvironment surrounding the Trp residues during binding. Interestingly, circular dichroism and Fourier transform infrared studies showed a significant change in the secondary structure of OVA upon binding with daphnetin. The efficacy of daphnetin in inhibiting protein fibrillation was confirmed through thioflavin T and Congo Red binding assays along with fluorescence microscopic imaging analysis. The thermodynamic assessment showed positive ΔH° [+(29.34 ± 1.526) kJ mol–1] and ΔS° [+(181.726 ± 5.465) J mol–1] values, indicating the presence of the hydrophobic forces, while negative ΔG° signifies spontaneous binding interactions. These experimental findings were further correlated with computational analysis, revealing daphnetin dynamics within the binding site of OVA.

In order to better understand the bioavailability and biocompatibility of polyphenol-assisted surface-modified bioengineered nanoparticles in nanomedicine applications, here, we address a series of photophysical experiments to quantify the binding affinity of serum albumin toward polyphenol-capped gold nanoparticles. For this, two different gold nanoparticles (AuNPs) were synthesized via the green synthesis approach, where curcumin and turmeric extract act as reducing as well as capping agents. The size, surface charge, and surface plasmon bands of the AuNPs were highly affected by the adsorption of human serum albumin (HSA) during protein corona formation, which was investigated using dynamic light scattering (DLS), ξ-potential, ultraviolet–visible (UV–vis) spectroscopy, and transmission electron microscopy (TEM) measurements. Fluorescence-based methods, absorbance, and SERS experiments were carried out to evaluate the binding aspects of AuNPs with HSA. We found that the AuNPs show moderate binding affinity toward HSA (Kb∼ 104M–1), irrespective of the capping agents on the surface. Hydrophobic association, along with some contribution of electrostatic interaction, played a key role in the binding process. The binding interaction was more toward the subdomain IIA region of HSA, as indicated by the competitive displacement studies using site-specific binders (warfarin and flufenamic acid). Because of the large surface curvature of small-sized AuNPs, the secondary structural conformations of HSA were slightly altered, as revealed by circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopy, and surface-enhanced Raman scattering (SERS) measurements. Additionally, the findings of the binding interactions were re-evaluated using molecular dynamics (MD) simulation studies by determining the root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration (Rg), and changes in the binding energy of HSA upon complexation with AuNPs. To determine the tentative evidence for pharmacokinetic administration, these biocompatible AuNPs were applied to inhibit the amyloid fibril formation of HSA and monitored by using the thioflavin T (ThT) assay, ANS fluorescence assay, fluorescence microscopic imaging, and FESEM. AuNPs were found to show better resistance toward fibrillation of the adsorbed protein.

AbstractMultispectroscopic and computational methods of exploring the interaction between a carrier protein and therapeutic compounds provide a preliminary investigation into establishing the efficacy of such compounds. Here, two coumarin derivatives, 7-hydroxycoumarin (7-HC) and 4-methyl-7-hydroxycoumarin (4-Me-7-HC), were selected to carry out numerous biophysical interaction studies with a model carrier protein, hen egg white lysozyme (HEWL). Fluorescence spectroscopy studies conducted between HEWL and 7-HC/4-Me-7-HC revealed the binding constants (Kb) were in the range of 104M−1, indicating a moderate nature of binding. The quenching mechanism observed during complexation process was an unusual static quenching due to the effect of temperature on the rate constant. Thermodynamic parameters revealed a positive ΔHand ΔSfor HEWL-7-HC/4-Me-7-HC, indicating hydrophobic forces played a dominant role in the interaction process. FRET studies suggested a possible non-radiative energy transfer from the donor (HEWL) to the acceptor (coumarins). Molecular docking studies revealed the interaction of 7-HC/4-Me-7-HC with intrinsic fluorophores, Trp63 and Trp108, Trp108 being an essential residue for binding as proven by molecular dynamic (MD) simulation. MD simulation studies also indicated conformational stability gained by HEWL upon interaction with 7-HC and 4-Me-7-HC. The microenvironment surrounding the Trp residues showed a significant Stoke’s shift on carrying out 3-D fluorescence. CD studies revealed a decrease in the alpha helical content of HEWL upon interacting with the ligands. Enzymatic assay conducted for HEWL in the presence of 7-HC/4-Me-7-HC saw an increase in the activity of HEWL, suggesting a change in structural conformation and stability of the protein, altering its activity.Communicated by Ramaswamy H. Sarma

In this study, we investigated the complexation of bioactive isoflavones, specifically biochanin A (BCA) and genistein (GEN), with hen egg white lysozyme (HEWL) and explored their inhibitory effects on HEWL modification using a combination of multi-spectroscopic and computational methods. The observed binding affinity was of a moderate nature (on the order of 104M−1), and a static quenching mechanism was identified in the fluorescence quenching process. Notably, the binding constant (Kb) for GEN (4.449 ± 0.262 × 104M−1) was found to be higher than that for BCA (3.707 ± 0.108 × 104M−1) towards HEWL. Our spectroscopic measurements, complemented by molecular docking calculations, suggested the involvement of Trp62 in the binding site of the isoflavones within the geometry of HEWL. The micro-environment surrounding the Trp-residues exhibited an increase in hydrophilicity, as indicated by Synchronous fluorescence (SFS) and three-dimensional fluorescence (3D) studies. Interestingly, circular dichroism (CD) studies revealed no marked alteration in the secondary structure of HEWL upon binding with the isoflavones. Furthermore, our investigation into the interaction patterns, employing FTIR and molecular docking studies, revealed a predominance of hydrogen bonding and hydrophobic interactions. Beyond the binding study, the isoflavones demonstrated a promising inhibitory effect on the d-ribose-mediated glycation of HEWL, as well as on HEWL fibrillation, as evidenced by fluorescence emission studies. Our findings not only exhibited an excellent correlation with experimental observations but also provided precise insights into the location and dynamics of isoflavones within the binding site through detailed analyses of molecular docking and molecular dynamics simulation data.

AbstractA new strain of a novel infectious disease affecting millions of people, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has recently been declared as a pandemic by the World Health Organization (WHO). Currently, several clinical trials are underway to identify specific drugs for the treatment of this novel virus. The inhibition of the SARS-CoV-2 main protease is necessary for the blockage of the viral replication. Here, in this study, we have utilized a blind molecular docking approach to identify the possible inhibitors of the SARS-CoV-2 main protease, by screening a total of 33 molecules which includes natural products, anti-virals, anti-fungals, anti-nematodes and anti-protozoals. All the studied molecules could bind to the active site of the SARS-CoV-2 protease (PDB: 6Y84), out of which rutin (a natural compound) has the highest inhibitor efficiency among the 33 molecules studied, followed by ritonavir (control drug), emetine (anti-protozoal), hesperidin (a natural compound), lopinavir (control drug) and indinavir (anti-viral drug). All the molecules, studied out here could bind near the crucial catalytic residues, HIS41 and CYS145 of the main protease, and the molecules were surrounded by other active site residues like MET49, GLY143, HIS163, HIS164, GLU166, PRO168, and GLN189. As this study is based on molecular docking, hence being particular about the results obtained, requires extensive wet-lab experimentation and clinical trials under in vitroas well as in vivoconditions.Communicated by Ramaswamy H. Sarma

AbstractDifferent post-translational changes in eye lens crystallin proteins contribute towards the development of cataract. We have studied in vitrooxidative modification of tryptophan (Trp) residues of human γD-crystallin (HGD) towards formation of N-formylkynurenine (NFK) associated with cataractogenesis. This oxidation was found to be inhibited by quercetin at relatively low concentration. Interactions between quercetin and HGD were further studied using fluorescence techniques. Binding and quenching constants were determined as ∼104M−1. Static quenching of fluorescence due to HGD–quercetin complex formation at ground state was confirmed by finding excited state life time of Trp residues. Energy transfer occurred between the protein and quercetin. Hydrogen bonding and/or van der Waals interactions were involved between HGD and quercetin. Synchronous and three-dimensional fluorescence along with far-UV CD studies suggested no major conformational alterations occurred in HGD due to quercetin binding. Experimental observations were supported by the docking results.Communicated by Ramaswamy H. Sarma

AbstractThe non-enzymatic glycation of plasma proteins by reducing sugars have important consequences on the conformational and functional properties of protein. The formation of advanced glycation end products (AGEs) is responsible for cell death and other pathological conditions. We have synthesized the glycated human serum albumin (gHSA) and characterized the same by using differential spectroscopic measurements. The aim of the present study is to determine the effect of glycation on the binding of human serum albumin (HSA) with bioactive flavonoid chrysin, which possesses anti-cancer, anti-inflammatory and anti-oxidant activities. The interaction of chrysin with HSA and gHSA was studied using multi-spectroscopic, molecular docking and molecular dynamics (MD) simulation techniques. Chrysin quenched the intrinsic fluorescence of both HSA and gHSA by static quenching mechanism. The value of the binding constant (Kb) for the interaction of HSA-chrysin complex (4.779 ± 0.623 × 105M−1at 300 K) was found to be higher than that of gHSA-chrysin complex (2.206 ± 0.234 × 105M−1at 300 K). Hence, non-enzymatic glycation of HSA significantly reduced its binding affinity towards chrysin. The % α-helicity of HSA was found to get enhanced upon binding with chrysin, and minimal changes were observed for the gHSA-chrysin complex. Site marker probe studies indicated that chrysin binds to subdomain IIA and IIIA of both HSA and gHSA. The results from molecular docking and MD simulation studies correlated well with the experimental findings. Electrostatic interactions followed by hydrogen bonding and hydrophobic interactions played major roles in the binding process. These observations may have some useful insights into the field of pharmaceutics.

AbstractThe interaction of 6-hydroxyflavone (6HF) with hen egg white lysozyme (HEWL) has been executed using multi-spectroscopic and computational methods. Steady state fluorescence studies indicated that static quenching mechanism is involved in the binding of 6HF with HEWL, which was further supported by excited state lifetime and UV–vis absorption studies. The binding constant (Kb) of the HEWL–6HF complex was observed to be 6.44 ± 0.09 × 104M−1at 293 K, which decreases with the increase in temperature. The calculation of the thermodynamic quantities showed that the binding is exothermic in nature with a negative enthalpy change (ΔH= −11.91 ± 1.02 kJ mol−1) along with a positive entropy change (ΔS= +51.36 ± 2.43 J K−1mol−1), and the major forces responsible for the binding are hydrogen bonding and hydrophobic interactions. The possibility of energy transfer from tryptophan (Trp) residue to the 6HF ligand was observed from Fo¨rster’s theory. The inclusion of 6HF within the binding site of HEWL induces some micro-environmental changes around the Trp residues as indicated by synchronous and three-dimensional (3D) fluorescence studies. The changes in secondary structural components of HEWL are observed on binding with 6HF along with a reduction in % α-helical content. Computational studies correlate well with the experimental finding, and the ligand 6HF is found to bind near to Trp 62 and Trp 63 residues of HEWL. Altogether, the present study provides an insight into the interaction dynamics and energetics of the binding of 6HF to HEWL.Communicated by Ramaswamy H. Sarma

Diabetes is a major problem in the world. The proteins became modified during glycation after reacting with the reducing sugars (e.g. D-glucose) via non-enzymatic pathways. The glycated analogue of human serum albumin (HSA) has been characterized with the help of multi-spectroscopic methods. It has been observed that six glucose molecules can bind covalently to HSA under experimental condition. The binding affinity of the modified HSA towards the dietary polyphenols has been estimated using UV–vis and fluorescence spectroscopic techniques. The binding constant values of the ligands were found to decrease after the modification of HSA.

Lactoferrin (LF) has several biological effects ranging from ribonuclease activity to antiangiogenic activity. It thus serves as a potential target protein for studies related to ribonucleolytic activity in association with its antiangiogenic activity. We have isolated buffalo LF and checked the ribonucleolytic activity via an agarose gel-based assay and precipitation assay. The ribonucleolytic activity of LF is lower compared to RNase A and the pH profile is a bell-shaped curve, with a pK1value of 5.43 and pK2of 7.65. The ribonuclease inhibitor that inhibits many ribonuclease-type proteins by forming a tight complex is unable to inhibit the ribonucleolytic property of LF. Fe(III) behaves as a noncompetitive inhibitor for the ribonucleolytic activity of protein. The superoxide-scavenging activity of the protein has also been measured. Histidine modification by diethylpyrocarbonate was monitored by UV–Vis spectroscopy at pH 7 and pH 8 and the effect towards the ribonucleolytic activity was determined. The antiangiogenic property of LF was investigated by the chorioallantoic membrane assay. Finally, the possible active site was analyzed via docking studies and correlated with the experimental study.

Green tea is rich in several polyphenols, such as (−)-epicatechin-3-gallate (ECG), (−)-epigallocatechin (EGC), and (−)-epigallocatechin-3-gallate (EGCG). The biological importance of these polyphenols led us to study the major polyphenol EGCG with human serum albumin (HSA) in an earlier study. In this report, we have compared the binding of ECG, EGC, and EGCG and the Cu(II) complexes of EGCG and ECG with HSA. We observe that the gallate moiety of the polyphenols plays a crucial role in determining the mode of interaction with HSA. The binding constants obtained for the different systems are 5.86 ± 0.72 × 104M−1(KECG-HSA), 4.22 ± 0.15 × 104M−1(KECG-Cu(II)-HSA), and 9.51 ± 0.31 × 104M−1(KEGCG-Cu(II)-HSA) at 293 K. Thermodynamic parameters thus obtained suggest that apart from an initial hydrophobic association, van der Waals interactions and hydrogen bonding are the major interactions which held together the polyphenols and HSA. However, thermodynamic parameters obtained from the interactions of the copper complexes with HSA are indicative of the involvement of the hydrophobic forces. Circular dichroism and the Fourier transform infrared spectroscopic measurements reveal changes in α-helical content of HSA after binding with the ligands. Data obtained by fluorescence spectroscopy, displacement experiments along with the docking studies suggested that the ligands bind to the residues located in site 1 (subdomains IIA), whereas EGC, that lacks the gallate moiety, binds to the other hydrophobic site 2 (subdomain IIIA) of the protein.

The binding of fisetin with human serum albumin (HSA) has been studied at different pH using UV-Vis, FTIR, CD and fluorescence spectroscopic techniques. The binding constants were found to increase with the rise in pH of the media. The negative H° (kJ mol-1) and positive S° (J mol-1 K-1) indicate that fisetin binds to HSA via electrostatic interactions with an initial hydrophobic association that result in a positive S°. In presence of potassium chloride (KCl) the binding constants were found to be decrease. The -helical content of HSA increased after binding with fisetin as analyzed from both CD and FTIR methods. The site marker displacement studies using fluorescence anisotropy suggest that fisetin binds to the hydrophobic pocket (Site 1, subdomain IIA) of HSA which is in good accordance with the molecular docking study. The change in accessible surface area (ASA) of residues of HSA was calculated to get a better insight into the binding.