Characterization of the denaturation of human alpha-lactalbumin in urea by molecular dynamics simulations

Molecular dynamics (MD) simulations were used to characterize the non-cooperative denaturation of the molten globule A-state of human alpha-lactalbumin by urea. A solvent of explicit urea and water molecules was used, corresponding to a urea concentration of approximately 6M. Three simulations were performed at temperatures of 293K, 360K and 400K, with lengths of 2 ns, 8 ns and 8 ns respectively. The results of the simulations were compared with experimental data from NMR studies of human alpha-lactalbumin and related peptides. During the simulations, hydrogen bonds were formed from the protein to both urea and water molecules as intra-protein hydrogen bonds were lost. Urea was shown to compete efficiently with water as both a hydrogen bond donor and acceptor. Radial distribution functions of water and urea around hydrophobic side chain atoms showed a significant increase in urea molecules in the solvation shell as the side chains became exposed during denaturation. A considerable portion of the native-like secondary structure persisted throughout the simulations. However, in the simulations at 360K and 400K, there were substantial changes in the packing of aromatic and other hydrophobic side chains in the protein, and many native contacts were lost. The results suggest that during the non-cooperative denaturation of the molten globule, secondary structure elements are stabilized by non-specific, non-native interactions.     

Structural details of urea binding to barnase: a molecular dynamics analysis.

BACKGROUND: The molecular mechanism of urea-induced protein unfolding has not been established. It is generally thought that denaturation results from the stabilizing interactions of urea with portions of the protein that are buried in the native state and become exposed upon unfolding of the protein. RESULTS: We have performed molecular dynamics simulations of barnase (a 110 amino acid RNase from Bacillus amyloliquefaciens) with explicit water and urea molecules at 300 K and 360 K. The native conformation was unaffected in the 300 K simulations at neutral and low pH. Two of the three runs at 360 K and low pH showed some denaturation, with partial unfolding of the hydrophobic core 2. The first solvation shell has a much higher density of urea molecules (water/urea ratio ranging from 2.07 to 2.73) than the bulk (water/urea ratio of 4.56). About one half of the first-shell urea molecules are involved in hydrogen bonds with polar or charged groups on the barnase surface, and between 15% and 18% of the first-shell urea molecules participate in multiple hydrogen bonds with barnase. The more stably bound urea molecules tend to be in crevices or pockets on the barnase surface. CONCLUSIONS: The simulation results indicate that an aqueous urea solution solvates the surface of a polypeptide chain more favorably than pure water. Urea molecules interact more favorably with nonpolar groups of the protein than water does, and the presence of urea improves the interactions of water molecules with the hydrophilic groups of the protein. The results suggest that urea denaturation involves effects on both nonpolar and polar groups of proteins.     

Non-covalent interaction of 2', 4', 5', 7'-tetrabromo-4, 5, 6, 7-tetrachlorofluorescein with proteins and its application

The interactions of 2', 4', 5', 7'-tetrabromo-4, 5, 6, 7-tetrachlorofluorescein (TBTCF) with BSA, ovalbumin (OVA) and poly-L-lysine (PLYS) at pH 3.70 have been investigated by combination of the spectral correction technique and the Langmuir isothermal adsorption. The active connection actions such as ion pairs, van der Waals' force, hydrogen bond, hydrophobic bond were proposed to explain the non-covalent interaction between TBTCF and BSA, OVA and PLYS. Effects of the electrolyte and high temperature indicated that union of the active connections between TBTCF and BSA and OVA was too firm to be destroyed. The relationship between the binding number of TBTCF and variety fraction of the amino acid residues was analyzed. The binding number of TBTCF depended on the number of positively charged amino acid residues. The other amino acid residues surrounded and seized TBTCF by hydrogen bonds and hydrophobic bonds when the electrostatic attraction pulled TBTCF to link protein. In addition, a novel method named the absorbance ratio difference was established for determination of protein in trace level and was applied with much higher sensitivity than the ordinary method.     

Effect of urea on peptide conformation in water: molecular dynamics and experimental characterization

Molecular dynamics simulations of a ribonuclease A C-peptide analog and a sequence variant were performed in water at 277 and 300 K and in 8 M urea to clarify the molecular denaturation mechanism induced by urea and the early events in protein unfolding. Spectroscopic characterization of the peptides showed that the C-peptide analog had a high alpha-helical content, which was not the case for the variant. In the simulations, interdependent side-chain interactions were responsible for the high stability of the alpha-helical C-peptide analog in the different solvents. The other peptide displayed alpha-helical unwinding that propagated cooperatively toward the N-terminal. The conformations sampled by the peptides depended on their sequence and on the solvent. The ability of water molecules to form hydrogen bonds to the peptide as well as the hydrogen bond lifetimes increased in the presence of urea, whereas water mobility was reduced near the peptide. Urea accumulated in excess around the peptide, to which it formed long-lived hydrogen bonds. The unfolding mechanisms induced by thermal denaturation and by urea are of a different nature, with urea-aqueous solutions providing a better peptide solvation than pure water. Our results suggest that the effect of urea on the chemical denaturation process involves both the direct and indirect mechanisms.     

Theoretical investigations on the hydrogen bonding of nitrile isomers with H2O,HF, NH3 and H2S

The hydrogen bonds formed by the interaction of nitriles with water, hydrogen fluoride, ammonia and hydrogen sulphide have been studied using B3LYP and second-order Møller–Plesset perturbation (MP2) methods and 6-311+ + G(d,p) basis set. The energies and structures of 80 hydrogen-bonded complexes between nitriles and small molecules were examined systematically using B3LYP and MP2 procedure. Categorisation of the hydrogen bonds involved in the various complexes led to an ordering of hydrogen bond donor and acceptor abilities for some functional groups. The interaction energies have been corrected for the basis set superposition error using Boy's counterpoise correction method. The Morokuma energy decomposition analysis reveals that the strong interactions are due to the attractive contributions from the electrostatic (ES), polarisation (PL) and charge transfer (CT) components. The topological parameters, electron density and Laplacian of electron density show excellent correlation with the hydrogen bond length. Natural bond orbital (NBO) analysis has also been performed to study the CT from proton acceptor to the antibonding orbital of the H–Y bond in the proton donor part of complexes. The frequency analysis of C–H…Y bond in the complexes indicates the blue-shifting nature largely in case of sp² hybridised carbon atom.     

Effects of low urea concentrations on protein-water interactions

Solvent properties of aqueous media (dipolarity/polarizability, hydrogen bond donor acidity, and hydrogen bond acceptor basicity) were measured in the coexisting phases of Dextran–PEG aqueous two-phase systems (ATPSs) containing .5 and 2.0 M urea. The differences between the electrostatic and hydrophobic properties of the phases in the ATPSs were quantified by analysis of partitioning of the homologous series of sodium salts of dinitrophenylated amino acids with aliphatic alkyl side chains. Furthermore, partitioning of eleven different proteins in the ATPSs was studied. The analysis of protein partition behavior in a set of ATPSs with protective osmolytes (sorbitol, sucrose, trehalose, and TMAO) at the concentration of .5 M, in osmolyte-free ATPS, and in ATPSs with .5 or 2.0 M urea in terms of the solvent properties of the phases was performed. The results show unambiguously that even at the urea concentration of .5 M, this denaturant affects partitioning of all proteins (except concanavalin A) through direct urea–protein interactions and via its effect on the solvent properties of the media. The direct urea–protein interactions seem to prevail over the urea effects on the solvent properties of water at the concentration of .5 M urea and appear to be completely dominant at 2.0 M urea concentration.     

C‐H…O hydrogen bonds in FK506‐binding protein–ligand interactions

Hydrogen bonds are important interaction forces observed in protein structures. They can be classified as stronger or weaker depending on their energy, thereby reflecting on the type of donor. The contribution of weak hydrogen bonds is deemed as an important factor toward structure stability along with the stronger bonds. One such bond, the C‐H…O type hydrogen bond, is shown to make a contribution in maintaining three dimensional structures of proteins. Apart from their presence within protein structures, the role of these bonds in protein–ligand interactions is also noteworthy. In this study, we present a statistical analysis on the presence of C‐H…O hydrogen bonds observed between FKBPs and their cognate ligands. The FK506‐binding proteins (FKBPs) carry peptidyl cis–trans isomerase activity apart from the immunosuppressive property by binding to the immunosuppressive drugs FK506 or rapamycin. Because the active site of FKBPs is lined up by many hydrophobic residues, we speculated that the prevalence of C‐H…O hydrogen bonds will be considerable. In a total of 25 structures analyzed, a higher frequency of C‐H…O hydrogen bonds is observed in comparison with the stronger hydrogen bonds. These C‐H…O hydrogen bonds are dominated by a highly conserved donor, the C^α/β of Val55 and an acceptor, the backbone oxygen of Glu54. Both these residues are positioned in the β4‐α1 loop, whereas the other residues Tyr26, Phe36 and Phe99 with higher frequencies are lined up at the opposite face of the active site. These preferences could be implicated in FKBP pharmacophore models toward enhancing the ligand affinity. This study could be a prelude to studying other proteins with hydrophobic pockets to gain better insights into ligand recognition. Copyright © 2013 John Wiley & Sons, Ltd.     

Theoretical study on the structures and properties of mixtures of urea and choline chloride

In this work, we investigated in detail the structural characteristics of mixtures of choline chloride and urea with different urea contents by performing molecular dynamic (MD) simulations, and offer possible explanations for the low melting point of the eutectic mixture of choline chloride and urea with a ratio of 1:2. The insertion of urea molecules was found to change the density distribution of cations and anions around the given cations significantly, disrupting the long-range ordered structure of choline chloride. Moreover, with increasing urea concentration, the hydrogen bond interactions between choline cations and Cl^? anions decreased, while those among urea molecules obviously increased. From the hydrogen bond lifetimes, it was found that a ratio of 1:2 between choline chloride and urea is necessary for a reasonable strength of hydrogen bond interaction to maintain the low melting point of the mixture of choline chloride with urea. In addition, it was also deduced from the interaction energies that a urea content of 67.7 % may make the interactions of cation–anion, cation–urea and anion–urea modest, and thus results in the lower melting point of the eutectic mixture of choline chloride and urea. The present results may offer assistance to some extent for understanding the physicochemical properties of the eutectic mixture of choline chloride and urea, and give valuable information for the further development and application of deep eutectic solvents.     

Structure,stability and water permeation of ([D-Leu-L-Lys-(D-Gln-L-Ala)3]) cyclic peptide nanotube: a molecular dynamics study

The structural stability of 8 × ([D-Leu-L-Lys-(D-Gln-L-Ala)₃]) cyclic peptide nanotube (CPN) in water and different phospholipid bilayers were explored by 100 ns independent molecular dynamics (MD) simulations. The role of non-bonded interaction energy between the side and main chains of cyclic peptide rings in different membrane environments assessed, wherein the repulsive electrostatic interaction energy between neighbouring cyclic peptide rings was found adequate to break hydrogen bond energy thereby to crumple CPN. Further, the water permeation across the CPN channel was studied in four types of phospholipid bilayers- DMPG (1,2-Dimyristoyl-sn-glycero-3-phosphorylglycerol), DMPS (1,2-Dimyristoyl-sn-glycero-3-phosphoserine), POPC (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPE (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) from MD simulations. DMPS membrane shows higher non-bonded interaction energies (?1913.06 kJ/mol of electrostatic interaction energy and ?994.13 kJ/mol of van der Waals interaction energy) with CPN due to the presence of polar molecules in lipid structure. Thusly, the non-bonded interaction energies were essential towards the stability of CPN than hydrogen bonds between the nearby cyclic peptides. The result also reveals the role of side chains, hydrogen bonds and non-bonded interaction energies in an aqueous environment. The diffusion coefficient of water obtained from means square deviation calculation shows similar coefficients irrespective of the lipid surroundings. However, the permeation coefficients demonstrate water flow in the channel relies upon the environment.     

Urea effects on protein stability: Hydrogen bonding and the hydrophobic effect

The effects of urea on protein stability have been studied using a model system in which we have determined the energetics of dissolution of a homologous series of cyclic dipeptides into aqueous urea solutions of varying concentration at 25°C using calorimetry. The data support a model in which urea denatures proteins by decreasing the hydrophobic effect and by directly binding to the amide units via hydrogen bonds. The data indicate also that the enthalpy of amide hydrogen bond formation in water is considerably higher than previously estimated. Previous estimates included the contribution of hydrophobic transfer of the α-carbon resulting in an overestimate of the binding between urea and the amide unit of the backbone and an underestimate of the binding enthalpy. Proteins 31:107–115, 1998. © 1998 Wiley-Liss, Inc.     

Differential ligand recognition by the Src and phosphatidylinositol 3-kinase Src homology 3 domains: circular dichroism and ultraviolet resonance Raman studies

Src homology 3 (SH3) domains recognize Pro-rich motifs using a hydrophobic cleft which contains several conserved aromatic residues. To investigate how aromatic residues contribute to ligand recognition, circular dichroism (CD) and 235 nm excited ultraviolet resonance Raman spectroscopies have been applied to Src and phosphatidylinositol 3-kinase (PI3K) SH3s. The CD analysis shows that Src SH3 binds to RPLPPLP (R-core) using aromatic residues with a dissociation constant (K(d)) of 10 microM. Moreover, intensity increases of the Trp and Tyr Raman bands suggest that the interaction is mediated by hydrophobic contacts and/or hydrogen bond formation with both Trp and Tyr residues. In the interaction of Src SH3 with VSLARRPLPPLP (VSL12) (K(d) 0.8 microM), Trp118 appears to form a strong hydrogen bond with VSL12, judging from significant intensity increases of the Trp Raman bands and the reported complex structure. In contrast, PI3K SH3 binds to R-core and VSL12 with lower affinities (K(d) 34 and 18 microM, respectively), and the interactions are suggested to be mediated mainly by hydrophobic contacts and/or hydrogen bond formation with Tyr residue(s). In the D21N mutant (Asp21 --> Asn) of PI3K SH3, whose hydrophobic cleft is deformed, Trp55 is shown to be responsible for the interaction with VSL12 by intensity increases of the Trp Raman bands. However, the affinity is severely decreased (K(d) 330 microM). These observations imply that SH3 domains associate with their ligands with distinct use of aromatic residues and that hydrogen bond formation with an SH3-conserved Trp residue in the well-ordered hydrophobic cleft is important for stable complex formation.     

Detection of interaction between lysionotin and bovine serum albumin using spectroscopic techniques combined with molecular modeling

A combination of fluorescence, UV–Vis absorption, circular dichroism (CD), Fourier transform infrared (FT-IR) and molecular modeling approaches were employed to determine the interaction between lysionotin and bovine serum albumin (BSA) at physiological pH. The fluorescence titration suggested that the fluorescence quenching of BSA by lysionotin was a static procedure. The binding constant at 298 K was in the order of 10⁵ L mol^?1, indicating that a high affinity existed between lysionotin and BSA. The thermodynamic parameters obtained at different temperatures (292, 298, 304 and 310 K) showed that the binding process was primarily driven by hydrogen bond and van der Waals forces, as the values of the enthalpy change (ΔH°) and entropy change (ΔS°) were found to be ?40.81 ± 0.08 kJ mol^?1 and ?35.93 ± 0.27 J mol^?1 K^?1, respectively. The surface hydrophobicity of BSA increased upon interaction with lysionotin. The site markers competitive experiments revealed that the binding site of lysionotin was in the sub-domain IIA (site I) of BSA. Furthermore, the molecular docking results corroborated the binding site and clarified the specific binding mode. The results of UV–Vis absorption, CD and FT-IR spectra demonstrated that the secondary structure of BSA was altered in the presence of lysionotin.     

Free energy simulations and MM-PBSA analyses on the affinity and specificity of steroid binding to antiestradiol antibody

Antiestradiol antibody 57-2 binds 17beta-estradiol (E2) with moderately high affinity (K(a) = 5 x 10(8) M(-1)). The structurally related natural estrogens estrone and estriol as well synthetic 17-deoxy-estradiol and 17alpha-estradiol are bound to the antibody with 3.7-4.9 kcal mol(-1) lower binding free energies than E2. Free energy perturbation (FEP) simulations and the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method were applied to investigate the factors responsible for the relatively low cross-reactivity of the antibody with these four steroids, differing from E2 by the substituents of the steroid D-ring. In addition, computational alanine scanning of the binding site residues was carried out with the MM-PBSA method. Both the FEP and MM-PBSA methods reproduced the experimental relative affinities of the five steroids in good agreement with experiment. On the basis of FEP simulations, the number of hydrogen bonds formed between the antibody and steroids, which varied from 0 to 3 in the steroids studied, determined directly the magnitude of the steroid-antibody interaction free energies. One hydrogen bond was calculated to contribute about 3 kcal mol(-1) to the interaction energy. Because the relative binding free energies of estrone (two antibody-steroid hydrogen bonds), estriol (three hydrogen bonds), 17-deoxy-estradiol (no hydrogen bonds), and 17alpha-estradiol (two hydrogen bonds) are close to each other and clearly lower than that of E2 (three hydrogen bonds), the water-steroid interactions lost upon binding to the antibody make an important contribution to the binding free energies. The MM-PBSA calculations showed that the binding of steroids to the antiestradiol antibody is driven by van der Waals interactions, whereas specificity is solely due to electrostatic interactions. In addition, binding of steroids to the antiestradiol antibody 57-2 was compared to the binding to the antiprogesterone antibody DB3 and antitestosterone antibody 3-C4F5, studied earlier with the MM-PBSA method.     

Biophysical and computational comparison on the binding affinity of three important nutrients to β-lactoglobulin: folic acid,ascorbic acid and vitamin K3

Small globular protein, β-lactoglobulin (βLG), which has significant affinity toward many drugs, is the most abundant whey protein in milk. In this study, the interaction of βLG with three important nutrients, ascorbic acid (ASC), folic acid (FOL), and vitamin K3 (VK3) was investigated by spectroscopic methods (UV–visible and fluorescence) along with molecular docking technique. The results of fluorescence measurements showed that studied nutrients strongly quenched βLG fluorescence in static (FOL and ACS) or static–dynamic combined quenching (VK3) mode. The values of binding constants (K_βLG-ASC ~ 4.34 × 10⁴ M^?1, K_βLG-FOL ~ 1.67 × 10⁴ M^?1and K_βLG-VK3 ~ 13.49 × 10⁴ M^?1 at 310 K) suggested that VK3 and FOL had stronger binding affinity toward βLG than ASC. Thermodynamic analysis indicated that hydrophobic interactions are the major forces in the stability of FOL–βLG complex with enthalpy- and entropy-driving mode while, hydrogen bonds and van der Waals interactions play a major role for βLG–ASC and βLG–VK3 associations. The results of 3D fluorescence FT-IR and UV–Visible measurements indicated that the binding of above nutrients to βLG may induce conformational and micro-environmental changes of protein. Also, there is a reciprocal complement between spectroscopic techniques and molecular docking modeling. The docking results indicate that the ASC, FOL, and VK3 bind to residues located in the subdomain B of βLG. Finally, this report suggests that βLG could be used as an effective carrier of above nutrients in functional foods.     

Theoretical study of noncovalent interactions in XCN···YO<Subscript>2</Subscript>H (X = F,Cl, Br,I; Y = P,As, Sb) complexes

Noncovalent interactions in XCN···YO₂H (X = F, Cl, Br, I; Y = P, As, Sb) complexes were investigated using ab initio calculations at the MP2/aug-cc-pVDZ level of theory. There are four different configurations of these complexes, and the complexes are formed via hydrogen bonds, halogen bonds, π-hole interactions, or dual interactions. An examination of binding distances and interaction energies suggested that π-hole bonds are more stable than the other interactions. Molecular electrostatic potentials, electron densities, second-order stabilization energies, and electron density differences were computed to study the character of these interactions.     

Molecular dynamics simulations of urea and thermal-induced denaturation of S-peptide analogue

Molecular dynamics simulations of the S-peptide analogue AETAAAKFLREHMDS in water at 278 and 358 K, and in 8 M urea at 278 K were performed. The results show agreement with experiments. The helix is stable at low temperature (278 K), while at 358 K, unfolding is observed. The effects of urea on protein stability have been studied. The data support a model in which urea denatures proteins by: (1) diminishing the hydrophobic effect by displacing water molecules from the solvent shell around nonpolar groups; and (2) binding directly to amide units (NH and CO groups) via hydrogen bonds. The results of cluster analysis and essential dynamics analysis suggest that the mechanism of urea and thermal-induced denaturation may not be the same.     

A study of the interactions between an IgG-binding domain based on the B domain of staphylococcal protein a and rabbit IgG

The nonantigenic interaction between a recombinant immunoglobulin G (IgG)-binding protein based on the B domain of Protein A fromStaphylococcus aureus (termed SpA¹) and the Fc fragment of rabbit IgG has been investigated. The contribution to binding of four putative hydrogen bond contacts between SpA¹ and IgG-Fc were examined by the individual substitution of the residues in SpA¹ involved in these interactions by others unable to form hydrogen bonds. It was found that the most important of the hydrogen bonds involved Tyr 18 which, when replaced by Phe, resulted in a twofold decrease in IgG-binding affinity. The residues of SpA¹ proposed to make close, mainly hydrophobic, contacts with Fc were replaced by residues with potential electrostatic charge to establish the importance of the hydrophobic interaction in the complex. The IgG-binding affinities of the mutant proteins were compared to the wild-type protein by a competitive enzyme-linked immunosorbant assay. The replacement of individual hydrophobic residues by His generated a number of novel IgG-binding proteins with reduced binding affinity at pH 5.0 but which maintained strong binding affinities at pH 8.0. The elution profile of human IgG₁-Fc (Fc fragment of human IgG₁) from a column made from an immobilized two-domain mutant protein shows that the complex dissociates at a higher pH relative to that of the non-mutated protein thus offering favorable elution characteristics.     

Effects of temperature and concentration on the structure of ethylene oxide–propylene oxide–ethylene oxide triblock copolymer (Pluronic P65) in aqueous solution: a molecular dynamics simulation study

The effects of temperature and solution concentration on the structure of triblock polymeric surfactant (ethylene oxide)₁₉(propylene oxide)₂₉(ethylene oxide)₁₉ (Pluronic P65) have been investigated by fully atomistic molecular dynamics simulations. The Flory–Huggins interaction parameter χ, hydrogen bonding and molecular mobility in the aqueous solution of P65 were investigated covering a composition range of 0.1–0.73 (water weight fraction) and a temperature range of 273–373 K. The Flory–Huggins parameters indicated that propylene oxide (PO) segments became hydrophobic with the increase in temperature, whereas ethylene oxide (EO) segments remained hydrophilic, which caused the increase in repulsion between EO and PO segments. The intermolecular hydrogen bonds in P65 solution including water–water hydrogen bonds and water–P65 hydrogen bonds increased with the increase in solution concentration and decreased with the increase in temperature. The critical micellar temperature of Pluronic P65 predicted by Flory–Huggins interaction parameter χ and hydrogen bonding was in good agreement with experimental data.     

The dominant interaction between peptide and urea is electrostatic in nature: a molecular dynamics simulation study

The conformational equilibrium of a blocked valine peptide in water and aqueous urea solution is studied using molecular dynamics simulations. Pair correlation functions indicate enhanced concentration of urea near the peptide. Stronger hydrogen bonding of urea-peptide compared to water-peptide is observed with preference for helical conformation. The potential of mean force, computed using umbrella sampling, shows only small differences between urea and water solvation that are difficult to quantify. The changes in solvent structure around the peptide are explained by favorable electrostatic interactions (hydrogen bonds) of urea with the peptide backbone. There is no evidence for significant changes in hydrophobic interactions in the two conformations of the peptide in urea solution. Our simulations suggest that urea denatures proteins by preferentially forming hydrogen bonds to the peptide backbone, reducing the barrier for exposing protein residues to the solvent, and reaching the unfolded state.     

Preferential binding of fisetin to the native state of bovine serum albumin: spectroscopic and docking studies

We have investigated the binding of the biologically important flavonoid fisetin with the carrier protein bovine serum albumin using multi-spectroscopic and molecular docking methods. The binding constants were found to be in the order of 10⁴ M^?1 and the number of binding sites was determined as one. MALDI-TOF analyses showed that one fisetin molecule binds to a single bovine serum albumin (BSA) molecule which is also supported by fluorescence quenching studies. The negative Gibbs free energy change (?G°) values point to a spontaneous binding process which occurs through the presence of electrostatic forces with hydrophobic association that results in a positive entropy change (+51.69 ± 1.18 J mol^?1 K^?1). The unfolding and refolding of BSA in urea have been studied in absence and presence of fisetin using steady-state fluorescence and lifetime measurements. Urea denaturation studies indicate that fisetin is gradually released from its binding site on the protein. In the absence of urea, an increase in temperature that causes denaturation of the protein results in the release of fisetin from its bound state indicating that fisetin binds only to the native state of the protein. The circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopic studies showed an increase in % α-helix content of BSA after binding with fisetin. Site marker displacement studies in accordance with the molecular docking results suggested that fisetin binds in close proximity of the hydrophobic cavity in site 1 (subdomain IIA) of the protein. The PEARLS (Program of Energetic Analysis of Receptor Ligand System) has been used to estimate the interaction energy of fisetin with BSA and the results are in good correlation with the experimental findings.