Investigating binding dynamics of trans resveratrol to HSA for an efficient displacement of aflatoxin B1 using spectroscopy and molecular simulation

Spectroscopic studies

The binding and interactive mode of flavonoid trans-resveratrol with HSA was investigated before its use as a dislodging agent for HSA bound AFB₁. The binding behavior was studied using spectroscopic tools like fluorescence spectroscopy and UV–visible spectroscopy. UV-absorption spectroscopy was used to ascertain the structural changes induced by trans-resveratrol upon binding with HSA. The UV-absorption property of HSA is by virtue of its aromatic amino acids (Trp, Tyr, and Phe) that impart a strong UV absorption signal at 280 nm¹⁹. Nevertheless, trans-resveratrol shows the absorption signal at 319 nm, as shown in Fig. 2a. From the Fig. 2a, it is clear that in the presence of an increasing concentration of trans-resveratrol, the hyperchromic effect was observed at absorption maxima (λ_max) of HSA, coupled with bathochromic shift, suggesting structural alterations in the native structure of HSA and a ground state complex formation between trans-resveratrol and HSA²⁰. The absorption spectrum also gives a clue about the existence of static quenching between trans-resveratrol and HSA, since in static type of quenching, the absorption spectra of native protein changes in the presence of a ligand molecule, however in dynamic quenching, it remains unaffected^21,22. Fluorescence spectroscopy was performed to get insight into the binding and thermodynamics parameters associated with the interaction of trans-resveratrol with HSA. When excited at 280 nm, a strong fluorescence quenching was observed in the fluorescence emission spectrum of HSA in the presence of an increasing concentration of trans-resveratrol (0–14 µM) as shown in Fig. 2b. HSA consists of fluorophores, critical for the fluorescent property of the protein viz, Trp, Tyr, and Phe, where the major contribution is from Trp-214 residue²³. Quenching is accompanied by redshift suggesting structural and conformational alterations in the native structure of HSA in the presence of trans-resveratrol. Bathochromic shift is the result of the increase in the polarity around fluorophores in HSA in the presence of trans-resveratrol²⁴.

Stern–Volmer quenching constant (K_SV) for binding trans-resveratrol with HSA was calculated according to Eq. (1) ²⁵. There is linearity between the concentration of T-res (Q) and F₀/F, as shown in Fig. 3a, at 298.15, 303.15, and 308.15 K. With the rise in temperature, a decrease in the slope of the Stern–Volmer plot is observed, the binding between HSA and trans-resveratrol also destabilizes with the increase in the temperature, suggesting a static mode of fluorescence quenching and quashing the existence of dynamic quenching mechanism operating between HSA and trans-resveratrol²⁶. Nevertheless, from Fig. 3a, if we look at the F₀/F vs Q plot at 298.15 K, at a higher concentration of trans-resveratrol, the F₀/F vs Q plot is shifted towards y axis, envisaging a mixed type of fluorescence quenching mechanism at 298.15 K, however, the plot shows the complete straight line at 303.15 and 308.15 K. The major contribution is for static nature of fluorescence quenching since the K_SV value decreases with increase in the temperature Further confirmation of the static quenching mechanism was done by calculating the bimolecular quenching constant (K_q) using Eq. (3)²⁵ of the “Methods” section. The numerical value of K_q obtained at three different temperatures, as shown in Table 1, is greater than the maximum scattering collision constant whose value is 2 × 10¹⁰ M⁻¹ s⁻¹, thus confirming the existence of static quenching and ground-state complex formation between HSA and trans-resveratrol^24,27. In a protein and ligand interaction, three different natures of fluorescence quenching can be observed, static, dynamic, and mixed (both static and dynamic) quenching. These different kinds of quenching patterns can be differentiated based on the K_SV values obtained at different temperatures. Static quenching is marked by ground-state complex formation between ligand molecule and protein²⁸, followed by decreases in the K_SV values with an increase in the temperature. The decrease in the K_SV is the result of a decrease in the ligand and protein stability with the rise in the temperature. However, in the dynamic nature of fluorescence quenching, the K_SV value increases with the rise in the temperature^29,30. Dynamic quenching results from the collision of the fluorophore and the ligand molecule. The mixed nature of fluorescence quenching displays both the properties of static and dynamic quenching³¹.

The binding constant (K_b) was calculated at three different temperatures, viz 298.15, 303.15, and 308.15 K using Eq. (4)^25,32, mentioned in the “Methods” section. The plot of log (F₀ − F)/F versus log (Q) is shown in Fig. 3b. The plot is a straight line, and the value of K_b was calculated to be 6.36 ± 0.32 × 10⁷ M⁻¹ at 298.15 K. Such a high binding constant is an indication of strong binding affinity between HSA and trans-resveratrol. The values of K_b and number of binding sites (n) at three different temperatures are reported in Table 2.

The binding affinity of AFB₁ towards HSA has already been reported earlier by Tan et al.³³. AFB₁ binds to HSA in subdomain IIA with a binding constant ~ 10⁴ M⁻¹. The binding constant (K_b) for AFB₁– HSA system is lower than the HSA-T-res system.

The Gibbs free energy (ΔG), enthalpy change (ΔH), and entropy change (ΔS) are the essential thermodynamic parameters that portray the spontaneity and favorability of a chemical reaction. These thermodynamic values are calculated using Eqs. (6) and (7) of the “Methods” section. Figure 3c shows the lnK versus 1/T (K) plot, the intercept and slope of the plot were used in the calculation of ΔS and ΔH, which was calculated to be −257.67 cal mol⁻¹ K^–1 and −87.60 kcal mol⁻¹, respectively. The magnitude of both ΔS and ΔH is negative, signifying hydrogen bonding and van der Waals interaction as the major forces acting in HSA and trans-resveratrol complex stabilization. The principal forces acting between the protein and ligand molecule are hydrogen bonding and van der Waals interaction when ΔS < 0 > ΔH³⁴. We also calculated the value of ΔG for the HSA-T-res system that was found to be negative, suggesting a favorable and spontaneous process of trans-resveratrol binding with HSA. All the thermodynamic parameters are reported in Table 2.

Location of the binding site of trans-resveratrol in HSA using site markers displacement assay

Our molecule of interest, trans-resveratrol, binds to HSA with a much higher affinity as compared to AFB₁, hence possessing the potential to compete with AFB₁. We further explored the binding site of trans-resveratrol on HSA using site markers, warfarin, and ibuprofen. These two site markers are routinely used probe molecules to locate the binding site of a small molecule on protein³⁵. Most of the ligand binds to the protein at Sudlow’s site I (subdomain IIA) and Sudlow’s site 2 (subdomain IIIA). Warfarin binds to subdomain IIA, and ibuprofen binds to subdomain IIIA. The binding location of AFB₁ on HSA has already been studied by Tan et al. and Poor et al.^17,33, confirming Sudlow’s site 1 as the binding pocket of AFB₁ on HSA. If trans-resveratrol and AFB₁ share the same binding site on the protein molecule, trans-resveratrol, by virtue of its higher binding constant (K_b) for HSA than AFB_1, could easily displace the mycotoxin from the HSA and increase its availability in the body in free form rather than bound form. The percentage displacement was calculated from the plot of F₂/F₁ × 100 versus probe/HSA, obtained using Eq. (7) of the “Methods” section^36,37. From Fig. 4, it is inferred that the percentage displacement of trans-resveratrol by warfarin is more prominent than ibuprofen, which provides a clue that trans-resveratrol binds at the site in HSA where warfarin binds, suggesting subdomain IIA or Sudlow’s site 1 as the binding site of trans-resveratrol in HSA. Various studies on ligand and protein interactions have used site markers displacement assays as a reliable method for locating the binding site of the ligand of interest in protein molecules^38,39.

Circular dichroism measurement

Circular dichroism is a valuable tool for deciphering the conformational or secondary structure change in the protein induced by a ligand⁴⁰. It is a routinely used technique involved in ligand and protein interaction to investigate the nature of the binding between protein and molecules⁴¹. Interaction of chromophores in the protein molecule, in an asymmetric milieu, with the polarized light results in CD signals⁴². Peptide bonds absorb polarized light in the far UV–region⁴³. The far UV-CD signal of HSA with predominant alpha helix exhibits two negative ellipticity at 208 and 222 nm as a consequence of n → π* and π → π* transition⁴⁴. From Fig. 5a, it is observed that the native HSA exhibited two peaks at 208 and 222 nm, suggesting the predominance of the alpha helix. The MRE value at 208 nm and percentage alpha helix was calculated using Eqs. (8) and (9) of the “Methods” section^38,45.

Native HSA showed 61.39% alpha-helical content. In the presence of trans-resveratrol (10 and 20 µM), the alpha-helical content was changed to 65.07 and 68.24%, respectively. Figure 5a,b indicate trans-resveratrol induced conformational changes and increase in alpha-helix in HSA, suggesting stabilization of native structure of HSA in the presence of trans-resveratrol. Lower concentrations of trans-resveratrol (2–8 µM) induced insignificant changes in the secondary structure of HSA. Figure 5b shows the CD values at 208 and 222 nm for HSA in the absence and presence of trans-resveratrol, which depicts a clear picture of the increase in CD (mdeg) values corresponding to increase in an alpha helix at 10 and 20 µM trans-resveratrol. Some phytochemicals and drug molecules, on interaction with albumins, have shown to increase the alpha helix and thereby its stability, and it is due to an increase in the extent of hydrogen bonding in the protein molecules^46,47,48.

Thermal stability of HSA in the presence of trans-resveratrol was also investigated using CD spectroscopy, by measuring changes in CD signal at 222 nm by sigmoidal fitting, as a function of temperature (20–90 °C). Hydrophobic interactions are the major contributors to the folding mechanism of protein. However, other factors like hydrogen bonding and electrostatic interactions also play significant roles in stabilizing protein structure⁴⁹. T_m is the midpoint transition temperature at which the equilibrium is maintained between folded and unfolded form⁵⁰. The thermal stability of the protein is directly proportional to its T_m value. In other words higher the T_m value more is the thermal stability of the protein⁵¹. The native HSA exhibited T_m values of 63.75 °C. However, in the presence of trans-resveratrol, it was increased to 66.25 °C. The increase in the T_m value from 63.75 °C to 66.25 °C confirms the trans-resveratrol-assisted folding of HSA. The thermal unfolding experiment further explores that the thermal stability of HSA is increased in the presence of trans-resveratrol. Certain drugs which bind to subdomain IIA, like warfarin and virstatin have been known to increase the T_m of HSA, as reported in earlier studies^52,53. Figure 6 shows the melting profile of HSA in the presence of trans-resveratrol.

Far UV- three dimensional CD spectra of HSA in the absence and presence of trans-resveratrol as a function of temperature were also plotted to envisage the ligand-induced structural and conformational perturbation of HSA at each temperature ranging from 20 to 90 °C. From Fig. 7, it is clear that with each rise in temperature from 20 to 90 °C, the negative ellipticity at 208 and 222 nm decreases, suggesting the unfolding of HSA as a function of temperature. In the presence of 20 µM trans-resveratrol, the alpha-helical content of HSA is protected at each rise in temperature, suggesting the trans-resveratrol mediated stabilization of the secondary structure of native HSA. From Table 3, it is evident that at 20 °C, native HSA showed 61.30% alpha-helix, and at 90 °C, it was reduced to 27.69% as a result of unfolding and temperature-induced denaturation. However, trans-resveratrol bound HSA at 20 °C exhibited 67.58%, higher than the HSA alone, at each increment in the temperature; HSA bound trans-resveratrol showed higher alpha-helix as compared to HSA alone at the same temperature. The drastic decrease in the alpha helix in HSA as a function of temperature is attributed to the reduction in the hydrogen bonding in the amino acids. Nevertheless, trans-resveratrol proved to be effective in protecting the unfolding of HSA, thereby restoring hydrogen bonding in the amino acid residues at a given temperature range. Figure 7 reflects the far UV-CD spectra of HSA in the presence of trans-resveratrol from 20 to 90 °C.

Displacement of AFB₁ from HSA by trans-resveratrol

After confirming that AFB₁ and trans-resveratrol shares the same binding site, subdomain IIA in HSA, displacement assay was followed to check the dislodging potential of trans-resveratrol against AFB₁ competing for the same binding pocket. In Fig. 8a, it is shown that each increasing concentration of trans-resveratrol (0–20 µM), displaces the AFB₁ bound to HSA from the HSA-AFB₁ system. However, when the displacing potential of AFB₁ against trans-resveratrol was studied for the HSA-Tres system, as shown in Fig. 8b, AFB₁ failed to dislodge trans-resveratrol from HSA, indicating the inability of AFB₁ to compete for albumin sharing the same binding site with trans-resveratrol. From Fig. 8b, it is clear that the increasing concentration of AFB₁ (0–20 µM) has no effect on the percentage displacement of trans-resveratrol bound to HSA.

Comparative analysis of the binding potentials of trans-resveratrol and AFB₁ to HSA

We also performed the comparative analysis of the effect of AFB₁ and trans-resveratrol on HSA, as depicted in Fig. 9. From the figure, based on the values of the fluorescence intensities, the emission spectrum of HSA underwent more quenching in the presence of trans-resveratrol than AFB₁. The decrease in the fluorescence intensity is more in HSA + T-res as compared to HSA + AFB₁. To further gain insights into the dislodging potential of trans-resveratrol, competing for HSA, equal concentrations of AFB₁ and trans-resveratrol were used for displacing trans-resveratrol bound HSA and AFB₁ bound HSA, respectively. From Table 4, it is evident that HSA alone (5 µM) showed fluorescence intensity of 47,776.6, the fluorescence intensity in the presence of AFB₁ and trans-resveratrol was decreased to 37,322.6 and 24,784.8, respectively. The fluorescence intensity of HSA for the HSA + T-res + AFB₁ system (Table 4) insignificantly differed from the fluorescence intensity of the HSA + T-res system, thereby confirming the inability of AFB₁ to displace trans-resveratrol from HSA-T-res. Nevertheless, the fluorescence emission intensity of HSA for the HSA + AFB₁ + T-res system (Table 4) significantly changed from the fluorescence emission intensity of the HSA + AFB₁ system, elucidating the potentiality of trans-resveratrol to dislodge the AFB₁ bound to albumin and apprehending its binding site.

Molecular docking and amino acid substitution studies

Molecular docking is a powerful computational approach to investigate ligand binding to the protein molecule at the atomic level⁵⁴. These in silico tools were used to explore and recognize the binding site of trans-resveratrol in HSA, corroborate the findings of spectroscopic studies, and get insight into the amino acid residues involved in the binding of trans-resveratrol with HSA. Figure 10 shows the best-docked pose and Sudlow’s site 1 as the binding pocket for the binding of trans-resveratrol with HSA. The binding energy for the interaction as calculated from the docking is −7.76 kcal mol⁻¹. If we look at Fig. 10, Trp-214 is present in the binding pocket, and it has a role in the stabilization of HSA trans-resveratrol complex. The amino acid residues surrounding the trans-resveratrol molecule are shown in Table 5. Figure 11 depicts the 2D picture of the amino acid residues in the vicinity of the trans-resveratrol and the nature of bonds formed between them. The hydrogen bonding and van der Waals interactions are best illustrated by the 2D pot of the HSA and trans-resveratrol interaction. Previous studies on the interaction of AFB₁ with HSA also confirmed Sudlow’s site 1 as the binding pocket for AFB₁ using in silico approach¹⁷, and the amino acids residues involved in the binding are in very close proximity to that involved in HSA and trans-resveratrol binding in the same Sudlow’s site 1. The van der Waals + hydrogen bonding + desolvation energy for HSA and trans-resveratrol complex was −9.07 kcal/mol, much higher than their electrostatic energy of −0.19 kcal/mol, thus proposing hydrogen bonding and van der Waals interaction as the major forces stabilizing the HSA and trans-resveratrol complex. This further corroborates our thermodynamic findings. Since the binding affinity of trans-resveratrol is much higher than the AFB₁, trans-resveratrol is able to displace bound AFB₁ to HSA for competing the binding site. Similar binding sites (subdomain IIA) have also been reported in a study of the binding of oxyresveratrol to HSA³⁹.

We further explored the binding of mutated HSA with trans-resveratrol. In mutated HSA, Trp-214 was substituted with neutral amino acid glycine to elucidate the changes in the binding fashion of trans-resveratrol with mutant HSA as compared to non-mutated HSA. Glycine was substituted in place of Trp-214 because glycine, the simplest amino acid, is also neutral. The molecular docking study revealed that the complex of trans-resveratrol with mutant HSA was less stabilized than the non-mutated complex because the binding energy of the mutated HSA complex was less negative compared to the non-mutated complex. The binding energy of the complex and the amino acid residues involved in the stabilization of the mutated HSA-trans-resveratrol complex is shown in Table 5. Interestingly, the binding site of trans-resveratrol was located at the same subdomain IIA as it was with non-mutated HSA. However, the number of hydrogen bonds was reduced to 2 only, as shown in Fig. 12. Gly-214 was not involved in any kind of bonding with trans-resveratrol. A mutation study was also performed by substituting the hydrophobic amino acid valine in Trp-214. Surprisingly, the binding site of trans-resveratrol on HSA was different as compared to native non-mutated HSA, and HSA with Trp-214 substituted with Gly. The binding energy of the complex was also less negative. The study with mutated HSA was significant since it investigates the role of Trp-214 during the stabilized complex formation between HSA and trans-resveratrol. The substitution of Trp-214 with either Gly or Val did not result in the highly stabilized complex formation as compared to the non-mutated HSA. The binding of the mutated HSA complex with trans-resveratrol was less negative as compared to the native HSA. The amino acid residues that form the binding pocket for mutant HSA and trans-resveratrol interaction are shown in Table 5. Figure 12 shows the best-docked pose of binding of trans-resveratrol with mutated HSA. Table 5 represents the amino acid residues involved and binding energies in the formation of mutated HSA-trans-resveratrol complex.

Molecular dynamic simulation studies

Molecular dynamic (MD) simulation studies were carried out to get insights into the dynamic nature of trans-resveratrol binding to HSA over the simulated period up to 100 ns. For the investigation of the system stability, root mean square deviations (RMSD) values of HSA backbone (C–Cα–N) and HSA-T-res complex were calculated, and the graph was plotted from 0 to 100 ns. From Fig. 13a, it is clear that the RMSD value of HSA alone was stable from 0 to 40 ns, and it was increased from 40 to 60 ns and later became stable again from 60 to 100 ns. For HSA-T-res complex, the RMSD value steadily increases from 0 to 20 ns. From 40 to 100 ns, the system becomes stabilized, and no significant increase or decrease in the RMSD value is obtained, and the system reaches the equilibrium suggesting HSA and trans-resveratrol is steadily bound to HSA at subdomain IIA. During the simulated time of 100 ns, the RMSD value of HSA and trans-resveratrol complex was lesser than the RMSD value of HSA alone, this also suggests the stability of the HSA system after binding with trans-resveratrol molecule. The RMSD values of HSA obtained in the present study are in good agreement with the previously reported studies⁵⁵.

Root mean square fluctuation (RMSF) values for HSA alone and HSA-trans-resveratrol complex were also evaluated to explore the protein flexibility⁵⁶. Figure 13b shows the plot of RMSF values versus amino acid residues in HSA. It is inferred that the amino acid residues in subdomain IIA are more rigid than other regions due to the complex formation between HSA and trans-resveratrol. Owing to the presence of random coils at the end of helix, flexibility is observed at the end residues, as shown in Fig. 13b.

Radius of gyration (R_g) value for HSA alone and HSA-trans-resveratrol complex was calculated to investigate the compactness of the protein and structural dynamics of the system over the simulated period of 100 ns. From Fig. 14, it is evident that the R_g value of HSA alone initially fluctuates at around 2.80 nm and reaches a maximum fluctuation value of 2.85 nm at around 50 ns. After that, it decreases continuously up to 90 ns, and after that, it remains constant up to 100 ns. The R_g values of HSA obtained are in accordance with the previously reported studies^55,57. On the other hand, the R_g value of HSA-trans-resveratrol complex initially decreases from 0 to 20 ns. Then it increases up to 30 ns, becomes stable, and reaches equilibrium from 30 to 100 ns. Figure 15 shows the dynamic nature of binding of trans-resveratrol to HSA at 0, 80, and 100 ns time at subdomain IIA. The findings of MD simulation studies corroborate the findings of our site markers displacement assays that also suggest subdomain IIA as the binding site of trans-resveratrol.