Complexation of Hen Egg White Lysozyme With Bioactive Phenolic Acids: Molecular Insights Into the Binding Mechanisms and Inhibitory Effects Against Protein Aggregation

Abstract

Protein misfolding and aggregation are central to many protein aggregation disorders, highlighting the need for effective aggregation inhibitors. This study investigated fructose‐induced aggregation of hen egg‐white lysozyme (HEWL) and its modulation by the phenolic acids ferulic acid (FA), caffeic acid (CA) and chlorogenic acid (CGA) at 100 and 200 μM. Circular dichroism and ThT fluorescence assays showed that fructose promoted aggregation through increased β‐sheet content and fluorescence intensity, respectively, whereas all phenolic acids suppressed β‐sheet formation. CA displayed the highest aggregation inhibition activity, followed by FA and CGA. FESEM imaging confirmed fructose‐induced HEWL aggregation without fibril formation and demonstrated disruption of aggregates in the presence of phenolic acids. These findings were explained by other biophysical characterisation studies. Fluorescence spectroscopy indicated a static quenching mechanism for all complexes, with FA and CA displaying atypical quenching behaviour. Binding constants suggested moderate affinity, with CA (4.11 ± 0.57 × 10⁴ M⁻¹ at 300 K) showing the strongest interaction. +ΔS and +ΔH indicated hydrophobic interactions as the main stabilising force for all complexes, while computational studies revealed additional H‐bonding interactions within aggregation‐prone regions (K‐peptide) of HEWL. Together, experimental and computational findings demonstrate that these phenolic acids effectively modulate HEWL aggregation by stabilising native‐like conformations and disrupting aggregate formation.

The graphical abstract illustrates the interaction of hen egg white lysozyme (HEWL) with ferulic acid, caffeic acid and chlorogenic acid. Integrated spectroscopic, computational and aggregation studies revealed protein–ligand binding, conformational modulation and inhibition of glycation‐induced HEWL aggregate formation, highlighting the aggregate inhibiting potential of these phenolic acids.

1. Introduction

Proteins must adopt and maintain their native three‐dimensional (3D) structures to perform their biological functions. Although cellular quality‐control systems continuously monitor protein folding and eliminate damaged proteins, failures in these mechanisms can result in the accumulation of partially unfolded or misfolded species [1]. These structurally destabilised proteins may undergo a nucleation‐dependent aggregation process, in which monomeric proteins first assemble into transient oligomeric intermediates that subsequently elongate into highly ordered amyloid fibrils enriched in cross‐β‐sheet structures (Figure S1) [2]. The resulting amyloid aggregates are often cytotoxic and have been implicated in numerous disorders, including Alzheimer's disease, Parkinson's disease, Creutzfeldt–Jakob disease, type II diabetes mellitus and systemic amyloidosis.

Among the various factors implicated in protein misfolding and aggregation, non‐enzymatic glycation has emerged as an important contributor to amyloidogenesis [2]. Glycation occurs through the reaction of reducing sugars or reactive carbonyl compounds with nucleophilic amino acid residues, leading to the formation of advanced glycation end products (AGEs) [3, 4]. These modifications can alter protein conformation, promote intermolecular cross‐linking, increase structural instability and facilitate the conversion of native protein structures into β‐sheet‐rich aggregates. Notably, glycated proteins are frequently detected within amyloid deposits, suggesting a close relationship between glycation and amyloid formation [5]. Consequently, the identification of compounds capable of preventing glycation‐induced protein aggregation has attracted considerable attention.

Although amyloid fibrils were initially associated primarily with neurodegenerative disease‐related proteins, extensive studies have demonstrated that many globular proteins can undergo amyloid‐like aggregation under suitable conditions [6]. Lysozyme represents one of the most extensively studied model proteins for investigating protein misfolding and fibrillation. Human lysozyme is directly associated with familial lysozyme systemic amyloidosis, a hereditary disorder characterised by widespread amyloid deposition in multiple organs [7, 8]. Amyloidogenic mutations destabilise the native structure of lysozyme, promoting the formation of partially folded intermediates that readily convert into cross‐β‐sheet‐rich fibrils [9]. Hen egg white lysozyme (HEWL) (Figure S2a) shares approximately 60% sequence homology and a highly conserved tertiary structure with human lysozyme, making it a widely accepted model for studying the molecular mechanisms of lysozyme aggregation and amyloid formation [10]. Natural polyphenols have attracted considerable interest as anti‐amyloid agents due to their ability to interact with proteins through H‐bonding, hydrophobic interactions and aromatic stacking interactions. These interactions can stabilise native protein conformations, remodel aggregation‐prone intermediates and inhibit fibril growth [11, 12]. Among polyphenolic compounds, phenolic acids (PA) such as ferulic acid (FA), caffeic acid (CA) and chlorogenic acid (CGA) (Figure S2b–d) are abundant dietary antioxidants that possess well‐documented anti‐glycation, anti‐oxidant and protein‐binding properties. However, despite increasing evidence supporting the anti‐amyloid activity of polyphenols, the molecular basis by which these PAs modulate glycation‐induced lysozyme aggregation remains insufficiently understood.

Therefore, the present study investigated the ability of FA, CA and CGA to inhibit glycation‐induced aggregation of HEWL, using fructose. In addition to evaluating their aggregation‐inhibiting activity using spectroscopic and microscopic techniques, detailed protein–ligand interaction studies were performed to elucidate the binding mechanisms, structural perturbations and thermodynamic characteristics governing complex formation. Experimental observations were further supported through molecular docking and molecular dynamics (MD) simulations to establish structure–activity relationships underlying the anti‐aggregation properties of these tested compounds.

2. Results and Discussion

2.1. Drug‐Likeliness and Physiochemical Properties of PAs

Drug‐likeness is commonly predicted using Lipinski's rule of five, which states that compounds with molecular weight <500, logP < 5, fewer than five H‐bond donors and fewer than 10 H‐bond acceptors generally show good absorption [13]. SwissADME (www.swissadme.ch) and reported literature were used to evaluate the physicochemical and Lipinski parameters of the PAs [14, 16]. FA and CA satisfied all Lipinski criteria, whereas CGA showed one violation (Table S1). The Egan and BOILED‐Egg models predicted absorption behaviour based on lipophilicity and polarity. FA was located in the yolk region, suggesting potential blood–brain barrier permeability, while CA fell within the white region, indicating good gastrointestinal absorption. However, CGA lay outside the ellipse, suggesting low absorption (Figure S3).

2.2. Aggregate Inhibition Properties of PAs

The glycation‐induced aggregation inhibitory effects of PAs (100 and 200 μM) were evaluated over 0–240 h using Thioflavin T (ThT) assay, a fluorescent probe specific for amyloid aggregates [17]. Fructose‐treated HEWL exhibited a progressive increase in ThT fluorescence intensity (Figure 1), indicating continuous β‐sheet formation during incubation [18]. A similar increase in ThT fluorescence has been reported for fructose‐induced fibrillation of human serum albumin, where glycation promoted α‐helix to β‐sheet transitions and subsequent fibril formation [19]. We assumed this could be a possibility in our observation, as proven by CD studies (discussed below). Contrastingly, fructose‐treated HEWL in the presence of FA, CA and CGA exhibited significantly lower intensities throughout the incubation period, demonstrating their ability to suppress β‐sheet formation. The inhibitory effect was observed for both concentrations (Figure 1). The ThT fluorescence spectra of all samples measured at 240 h are shown in Figure S4a.

FIGURE 1.

(a) Time‐dependent kinetic profiles of fructose‐treated HEWL in the absence and presence of ferulic acid (FA), caffeic acid (CA), and chlorogenic acid (CGA) at two concentrations (100 and 200 μM), monitored by ThT fluorescence assay. (b) Bar representation of the corresponding ThT fluorescence intensities at different incubation times. (c–e) Individual kinetic plots showing the inhibitory effects of FA, CA and CGA, respectively, on HEWL aggregation. HEWL was incubated in 50 mM phosphate buffer (pH 7.4) at 37 °C, and ThT fluorescence was recorded at the indicated time intervals. λ _ex (ThT) = 450 nm. Error bars represent the standard deviation from three independent experiments.

The far‐UV CD spectrum of a protein shows two negative peaks at 208 and 222 nm, associated with α‐helical content. The 208 nm band corresponds to the π–π* transition of α‐helices and the 222 nm band arises from the n–π* transition linked to α‐helix and coils. This method was used to assess secondary structural changes in fructose‐treated HEWL after 240 h of incubation with and without PAs. A reduction in the CD ellipticity at 208 nm was observed, indicating a loss of α‐helical structure, which was more pronounced in samples incubated without PAs. However, the presence of PAs attenuated this structural transition. These findings suggested that the PAs effectively preserved the native α‐helical conformation of HEWL under aggregation conditions (Figure S5). Among the compounds tested, CA exhibited the strongest protective effect, demonstrating the highest retention of α‐helical content. These observations agree with the ThT results, confirming increased β‐sheet formation in fructose‐treated HEWL and the inhibitory effect of PAs on this structural transition.

FESEM images of fructose‐treated HEWL samples incubated for 240 h in the absence and presence of PAs were acquired to evaluate their morphological characteristics. The fructose‐treated protein exhibited predominantly globular and amorphous aggregated structures, with no formation of fibrils (Figure 2a). The samples incubated with PAs showed a noticeable reduction in these aggregated structures, indicating their ability to disrupt aggregation assemblies (Figure 2b–d). Among the PAs tested, CA demonstrated the greatest efficacy in reducing aggregates. These results are significant as they show that fructose cannot fibrillate HEWL after 10 days (240 h) but results in aggregate formation instead.

FIGURE 2.

FESEM images of aggregate formation of (a) HEWL, (b) HEWL + FA, (c) HEWL + CA and (d) HEWL + CGA captured after 240 h of incubation with fructose.

A one‐way ANOVA was performed to compare ThT fluorescence intensity among untreated HEWL and PA‐treated samples at the two different concentrations. The analysis revealed a highly significant effect of treatment on ThT fluorescence (p < 0.0001), indicating aggregate formation differed substantially across experimental conditions. These results indicate that all compounds reduce HEWL aggregation compared to the untreated control, confirming their potential to disrupt aggregation. The magnitude of inhibition varied with PA identity, with CA showing the strongest effect and CGA the weakest. The relatively small differences between 100 and 200 μM concentrations suggested that inhibitory activity was not strongly dose‐dependent within the tested range. To further evaluate whether the PA concentration used influenced anti‐aggregation activity, a two‐way ANOVA was performed using only the PA‐treated samples at 100 and 200 μM. The analysis revealed a significant effect of PA identity on ThT fluorescence intensity (p < 0.0001), indicating that the tested compounds differed in their ability to modulate HEWL aggregation. In contrast, concentration had no significant effect on ThT fluorescence (p > 0.05), suggesting limited dose dependence within the tested range. No significant PA–concentration interaction was observed (p > 0.05), indicating that the effect of concentration was similar across all compounds. From a mechanistic perspective, the observed differences in aggregate mitigation likely arise from variations in PA structure, including differences in aromaticity, H‐bonding capacity and hydrophobic interactions with HEWL aggregation‐prone regions. These interactions may influence early nucleation events or stabilise partially unfolded intermediates, thereby altering aggregate formation kinetics.

Other experiments were conducted after 240 h of glycation‐induced aggregation. Congo red (CR) dye binds to amorphous aggregates and causes a redshift in its absorbance spectra [20]. The spectra of native HEWL + CR and CR alone showed low absorbance between 400–600 nm (Figure S4b). In contrast, fructose‐treated HEWL + CR spectra showed increased absorbance around 500 nm, indicating CR binds to aggregates. However, in the presence of PAs, the absorbance intensity was reduced, suggesting these compounds partially inhibited aggregation. 8‐Anilino‐1‐naphthalene‐sulphonic acid (ANS), a probe that binds hydrophobic protein pockets, was used to assess HEWL hydrophobicity [21, 22]. ANS showed low fluorescence with native HEWL but increased intensity after aggregation, indicating exposure of hydrophobic regions (Figure S4c). In the presence of PAs, the intensity decreased, suggesting preservation of HEWL structural integrity by PAs, reducing accessibility of ANS to hydrophobic pockets. The turbidity of each sample was measured at 350 nm. The absorbance of aggregated HEWL was high but in the presence of PAs, the absorbance measured was lower as the turbidity of the solution decreased (Figure S6a). UV–vis spectra of HEWL and fructose‐treated HEWL in the absence and presence of PAs were recorded between 200–500 nm to monitor changes in aromatic residues absorbing at 280 nm. Fructose‐treated HEWL showed increased absorbance compared to native protein, indicating exposure of aromatic residues due to structural changes (Figure S6b). In the presence of PAs, reduced intensity suggested moderate preservation of HEWL structure. Fructose‐treated HEWL exhibited reduced Trp fluorescence and a redshift relative to untreated HEWL (Figure S6c), indicating increased exposure of Trp residues to the solvent and consequent quenching by water molecules [23]. In presence of PAs, further fluorescence quenching accompanied by a blueshift was observed, suggesting interactions between the PAs and the aromatic residues (Figure S6c) [24]. Similar findings have been documented [25, 26]. Overall, the results demonstrated that all PAs effectively attenuate HEWL aggregation. The study becomes significant when relating the PAs with their physicochemical properties. CA, the most promising candidate, along with its therapeutic properties, becomes more favourable due to its non‐toxicity under common intake conditions [27] and its bioavailability in the nanomolar range [28, 29]. FA, which followed closely, demonstrated predicted blood–brain barrier permeability, making it a significant candidate for further investigation in neuroprotective and anti‐amyloid therapeutic applications.

2.3. UV–Vis Absorption Studies

Figure S7 represents the UV–vis absorption spectra of native HEWL, HEWL + PAs and free PAs in 200–600 nm wavelength range. At 280 nm, a peak characteristic for HEWL was observed, which arises due to π–π* transitions of Trp and Tyr residues. An increase in absorbance was witnessed with the addition of PAs, indicating a successful interaction between the protein and the individual compounds. Furthermore, the difference absorption spectrum of HEWL was generated by subtracting the spectrum of HEWL + PA complex from that of the corresponding PA alone. The resulting spectrum showed no overlap with the spectrum of the native protein, indicating that a static quenching mechanism existed for all complexes. The finding suggested that an interaction occurred between HEWL and each PA due to the formation of ground‐state complexes.

2.4. Fluorescence Spectroscopy

Fluorescence spectroscopy provides valuable insight into protein–ligand interactions. The intrinsic fluorophores of HEWL exhibit maximum fluorescence at 344 nm upon excitation at 295 nm, with Trp62, Trp63 and Trp108 known as the intrinsic fluorophores. A gradual addition of PAs resulted in a decrease in fluorescence intensity, indicating quenching of Trp residues due to interaction with PAs (Figure 3). To distinguish between static and dynamic quenching mechanisms, temperature‐dependent fluorescence measurements were performed at 293, 300 and 307 K [30]. Fluorescence data were corrected for the inner filter effect (IFE) using Equation (1), as PAs absorb at both the excitation (295 nm) and emission (344 nm) wavelengths of HEWL [30, 31].

FIGURE 3.

Fluorescence emission spectra of the intrinsic fluorophore of HEWL complexed with increasing concentration of (a) FA, (b) CA and (c) CGA in 20 mM phosphate buffer. [HEWL] = 3 µM. [PAs] = 0–16.4 µM. λ _ex = 295 nm.

$$F_{\mathrm{corr}}=F_{\mathrm{obs}}\times {10}^{\left(\frac{A_{\mathrm{ex}}+A_{\mathrm{em}}}{2}\right)}$$ (1)

where F _corr and F _obs are the corrected and observed fluorescence intensities, respectively, and A _ex and A _em are the absorbance values of PAs at the excitation and emission wavelengths of HEWL, respectively.

The corrected fluorescence intensities were used to investigate the quenching mechanism and binding parameters. Using the classical Stern–Volmer equation (Equation (2)), the quenching constant (K _SV) value was determined for HEWL + FA/CA/CGA complexes [32, 33].

$$\frac{F_0}{F_{\mathrm{corr}}}=1+K_{\mathrm{SV}}\hspace{0.17em}\left[Q\right]=1+k_{\mathrm{q}}{\tau }_0\hspace{0.17em}\left[Q\right]$$ (2)

where F ₀ and F _corr are the fluorescence intensities in the absence and presence of PAs, respectively, after IFE correction. Q is the PAs concentration, k _q is the bimolecular quenching constant and τ ₀ is the average lifetime of the excited state fluorophore in the absence of PAs.

Stern–Volmer plots (Figure S8) displayed upwards curvature for all complexes, initially suggesting combined static and dynamic quenching. However, analysis using modified Stern–Volmer equations Equations (3) and (4) produced regression plots (Figure S9) with imaginary values calculated for K _s (static quenching constant) and K _D (dynamic quenching constant), indicating the presence of a single quenching mechanism [30].

$$\frac{F_0}{F_{\mathrm{corr}}}=(1+K_{\mathrm{D}}\hspace{0.17em}\left[Q\right])(1+K_{\mathrm{S}}\hspace{0.17em}\left[Q\right])$$ (3)

$$\left(\frac{F_0-F_{\mathrm{corr}}}{F_{\mathrm{corr}}}\right)\hspace{0.17em}\frac{1}{\left[Q\right]}=(K_{\mathrm{S}}+K_{\mathrm{D}})+K_{\mathrm{S}}{K}_{\mathrm{D}}\hspace{0.17em}\left[Q\right]$$ (4)

The linear portion of the Stern–Volmer plots was used to analyse the K _SV parameter. For HEWL + FA/CA, K _SV increased with temperature, indicating dynamic quenching, whereas HEWL + CGA showed a decrease, suggesting static quenching (Table S2). However, calculated k _q values (k _q = K _SV/τ ₀) were approximately 10³ times greater than the maximum diffusion‐controlled constant (2 × 10¹⁰ M⁻¹ s⁻¹), indicating that static quenching predominates in all complexes (Table S2) [34]. Under different temperature conditions, studies conducted by different research groups also saw similar observations [35].

The conflicting trends between K _SV and k _q for HEWL + FA/CA complexes were explained using Arrhenius theory. The rate constant depends on temperature, and static quenching is particularly sensitive to temperature changes [36, 37]. While k _q for dynamic quenching is limited to 2 × 10¹⁰ M⁻¹ s⁻¹ in aqueous media, no such limit exists for static quenching. Increasing temperature generally destabilises the ground–state complex, reducing quenching efficiency, i.e., k _q value decreases. However, reduced solvent viscosity at higher temperatures may increase collisions between FA/CA and HEWL, influencing the observed K _SV [38]. If the increase in K _SV outweighs the reduction caused by complex instability, an overall increase in K _SV may occur with a rise in temperature [38, 39]. Thus, the increase in both K _SV and k _q with temperature suggested an “unusual” static quenching mechanism for HEWL + FA/CA complexes [38, 40].

The activation energy (E _a) of the quenching process was calculated using the Arrhenius equation (Equation (5)) [41].

$$\ln \hspace{0.17em}\left(\frac{K_{\mathrm{SV}}}{{\tau }_0}\right)=\ln \hspace{0.17em}\left(k_{\mathrm{q}}\right)=-\frac{E_{\mathrm{a}}}{RT}+\ln A$$ (5)

where A is the pre‐exponential factor. A linear plot of  lnk _q versus 1/T (Figure S10) yielded $E_a$ values of +(12.15 ± 0.56) kJ mol⁻¹ for HEWL + FA and +(17.18 ± 0.65) kJ mol⁻¹ for HEWL + CA. These values exceed those reported for similar protein–ligand systems, indicating a strong temperature dependence and supporting the presence of an unusual static quenching [40, 42].

2.5. Binding Constant and Number of Binding Sites

The above results confirmed the interaction between HEWL and PAs. Assuming each PA binds independently to equivalent sites on HEWL, the binding constant (K _b) and number of binding sites (n) were determined using Equation (6) [43].

$$\log \frac{F_0-F_{\mathrm{corr}}}{F_{\mathrm{corr}}}=n\hspace{0.17em}log{K}_{\mathrm{b}}-n\hspace{0.17em}\log \hspace{0.17em}\left[\frac{1}{\{\left[Q\right]-\frac{\left[P_{\mathrm{t}}\right]\hspace{0.17em}(F_0-F_{\mathrm{corr}})}{F_0}\}}\right]$$ (6)

where P _t represents the total protein concentration.

The double logarithm plot of log(F ₀− F _corr)/F _corr versus log[1/{[Q] − (F ₀ − F _corr)[P _t]/F ₀}] (Figure 4) yielded the n and K _b values (Table 1). The n values were found to be greater than 1, which could be due to multiple binding sites or a higher quencher concentration around the fluorophore. This could explain the observed non‐linear Stern–Volmer curves. K _b values were in the range of 10⁴ M⁻¹, as previously reported [44, 46]. This was a moderate association compared to other protein‐ligand complexes with stronger binding associations of 10⁶–10⁸ M⁻¹ range [47]. The order of association observed is critical for reversibility in the binding between the protein and PAs. The K _b value was noted to increase with an increase in temperature for all complexes, which suggested that they were stabilised as the temperature rose (Table 1).

FIGURE 4.

Double‐log binding plots of log(F ₀− F _corr)/F _corr versus log[1/{[Q] − [P _t](F ₀− F _corr)/F ₀}] of HEWL complex with (a) FA, (b) CA and (c) CGA at three different temperatures.

TABLE 1.

The binding constant (K _b) and number of binding sites (n) of HEWL complexed with PAs.

Complex	Temperature, K	K b , M−1 × 104	n
HEWL + FA	293	3.64 ± 0.07	1.55 ± 0.01
	300	4.06 ± 0.07	1.38 ± 0.02
	307	4.64 ± 0.06	1.57 ± 0.03
HEWL + CA	293	3.79 ± 1.20	1.35 ± 0.02
	300	4.11 ± 0.57	1.20 ± 0.02
	307	5.56 ± 1.01	1.62 ± 0.10
HEWL + CGA	293	3.42 ± 0.69	1.25 ± 0.03
	300	3.88 ± 0.45	1.19 ± 0.02
	307	4.36 ± 0.46	1.41 ± 0.05

To validate the K _b values observed for all complexes, the molecular structure of each PA was scrutinised. FA is a cinnamic acid consisting of a methoxy group on C‐3 and a hydroxy group on C‐4 on the phenyl ring. CA consists of hydroxy substituents on C‐3 and C‐4 on the phenyl ring, and CGA is the ester formed between CA and quinic acid. CA displayed the highest K _b value, which could be correlated to the presence of two hydroxyl groups. From docking studies, HEWL + CA two dimensional (2D) plots (Figure 7e), the C‐3 and C‐4 hydroxyl groups formed three H‐bonds with Trp108 and Gln57. An additional H‐bond was observed between the carboxyl group and Asn103. The presence of four H‐bonds provided enhanced stability to the complex, which could explain the higher K _b value observed compared to HEWL + FA/CGA complexes, which showed fewer H‐bonds (Figure 7b,h). Similar observations were made based on the 2D plots generated from MD simulation (Figure S20a–c). The presence of a methoxy group on FA could have also provided steric hindrance, resulting in a lower K _b value [48]. The lowest K _b value observed for HEWL + CGA could be due to the presence of the quinic moiety, preventing it's penetration into the active site of HEWL.

FIGURE 7.

Illustration of docked poses of HEWL complexed with (a) FA, (d) CA and (g) CGA, along with their 2D plots (b) FA, (e) CA and (h) CGA. Surface hydrophobicity of the binding site of (c) FA, (f) CA and (g) CGA within HEWL. Scale: +3 (Hydrophobic) to −3 (Hydrophilic).

2.6. Non‐Covalent Interactions

Non‐covalent interactions stabilise HEWL + PA complexes, including hydrophobic, H‐bonding, electrostatic and van der Waals forces [49]. These interactions were evaluated by determining the thermodynamic parameters (ΔH and ΔS) using the van’t Hoff Equation (7), while Gibbs free energy (ΔG) was calculated using Equation (8).

$$\log \hspace{0.17em}{K}_{\mathrm{b}}=\frac{-\text{Δ}H}{2.303RT}+\frac{\text{Δ}S}{2.303R}$$ (7)

$$\text{Δ}G=\text{Δ}H-T\text{Δ}S$$ (8)

van’t Hoff plots (1/T vs. log K _b) for HEWL + FA/CA/CGA (Figure S11) showed linear behaviour, indicating constant ΔH within the studied temperature range. The calculated thermodynamic parameters are listed in Table 2. Negative ΔG values confirmed that complex formation was spontaneous. Positive ΔH and ΔS values suggested that hydrophobic interactions dominate the binding process [49]. Although positive ΔH indicated an endothermic contribution, the large positive ΔS resulted in a favourable TΔS term, leading to overall spontaneity and stabilisation of the complexes. A comparison of thermodynamic parameters is presented in Figure S12.

TABLE 2.

Thermodynamic parameters for HEWL complexed with PAs determined from steady‐state fluorescence.

Ligands	Temp., K	ΔH, kJ mol−1	ΔS, J mol−1 K−1	ΔG, kJ mol−1
FA	293	+(8.87 ± 0.36)	+(117.98 ± 1.17)	−(25.69 ± 0.04)
	300			−(26.52 ± 0.04)
	307			−(27.06 ± 0.03)
CA	293	+(24.61 ± 0.15)	+(171.38 ± 0.17)	−(25.59 ± 0.11)
	300			−(27.78 ± 0.11)
	307			−(27.98 ± 0.11)
CGA	293	+(12.73 ± 0.11)	+(97.24 ± 0.54)	−(26.55 ± 0.02)
	300			−(27.02 ± 0.01)
	307			−(27.49 ± 0.03)

2.7. Microenvironmental Changes

2.7.1. Synchronous Fluorescence

Synchronous fluorescence spectra recorded at fixed wavelength differences (Δλ) of 15 or 60 nm provide information on changes in the polarity of aromatic residues upon ligand binding [50]. The interaction of PAs with HEWL resulted in a decrease in HEWL fluorescence at an offset wavelength of 60 nm (specific for Trp residues), along with a redshift in the presence of FA/CA and a blueshift in the presence of CGA (Figure 5). The redshift observed in the former complexes indicated the microenvironment around the Trp residues was becoming more polar, whereas the blueshift observed on interacting with CGA suggested decreasing polarity. When the offset wavelength was set to 15 nm (specific for Tyr residues), a reduction in fluorescence intensity was observed for all complexes without any shift in the emission maxima, suggesting negligible microenvironment transformation around the Tyr residues (Figure S13). The data have been provided in Table S3. The change in polarity of Trp residues in the presence of PAs was similarly noted [44, 46].

FIGURE 5.

Synchronous fluorescence spectra of HEWL in the presence of increasing concentrations of (a) FA, (b) CA and (c) CGA. A depiction of variation in the emission maxima (λ _max) at Δλ = 60 nm and Δλ = 15 nm with increasing concentration of the quencher (d) FA, (e) CA and (f) CGA. [HEWL] = 3 µM. [PAs] = 0–16.4 µM.

2.7.2. 3D Fluorescence

3D fluorescence spectroscopy was employed to examine conformational and microenvironmental changes in HEWL upon PAs binding [51]. The 3D spectra and corresponding contour maps of HEWL and its complexes are shown in Figure S14, with data summarised in Table S4. Four peaks were observed: peaks a and b correspond to first‐ and second‐order Rayleigh scattering. Peak 1, arising from aromatic residues of HEWL, [36, 52] showed a marked decrease in intensity upon PA binding. In addition, redshifts of 5 and 6 nm were observed for HEWL + FA and HEWL + CA, respectively, indicating structural changes that exposed aromatic residues to a more polar environment. In contrast, HEWL + CGA exhibited a 2 nm blueshift, suggesting movement of these residues towards a more non‐polar environment. These observations are consistent with synchronous fluorescence results. Peak 2, attributed to fluorescence from higher electronic states of Trp and Tyr [52], also showed reduced intensity and a noticeable Stokes shift in presence of PAs.

2.7.3. Red Edge Excitation Shift Studies (REES)

REES involves a shift in the emission maximum to longer wavelengths as the excitation wavelength approaches the red edge of the absorption band. This arises from electronic coupling between Trp indole rings and nearby dipoles in environments with slow solvent relaxation, making it useful for probing motions around Trp in proteins. Here, Trp was excited at two excitation wavelengths, 295 and 305 nm, to investigate the REES effect of HEWL in the absence and presence of PAs. The value of Δλ _em,max = (λ _{em, max} at 295 nm excitation wavelength) – (λ _em,max at 305 nm excitation wavelength) is presented in Table S5. Native HEWL displayed a REES effect of 3 nm. However, on complexing with FA/CA/CGA, a REES shift of 6/5/7 nm, respectively, was observed. The increased shift of the emission maxima in the presence of PAs suggested that the mobility of Trp was slightly minimised due to restrictions levied on it owing to the interactions.

2.8. Secondary Structural Changes

2.8.1. Circular Dichroism (CD)

The change in CD spectra of HEWL provided evidence of conformational changes of the protein induced by binding with PAs (Figure 6a–c). On increasing the concentration of PAs, a decrease in the intensity of 208 and 222 nm peaks, without any shifts, was observed. This indicated that the binding of PAs with HEWL induced secondary structural changes in the protein. The α‐helical content decreased from 21.16 ± 0.75% (native HEWL) to 15.86 ± 0.11%, 16.20 ± 0.45% and 12.16 ± 0.15% upon interacting with FA, CA and CGA, respectively. The decrease in α‐helical content was consistent with other studies [44, 46]. The percentage of other secondary structural components of HEWL and its complexes is tabulated (Table S6).

FIGURE 6.

CD spectra of HEWL in the absence and presence of (a) FA, (b) CA and (c) CGA in 20 mM phosphate buffer. [HEWL] = 20 µM. FT‐IR spectra of HEWL and HEWL complexed with (d) FA, (e) CA and (f) CGA in 20 mM phosphate buffer, in the range of 1800–1100 cm⁻¹. [HEWL:PAs] = 1:2 molar ratio.

2.8.2. Fourier Transform Infrared Spectroscopy (FT‐IR)

FT‐IR investigates secondary structural changes in HEWL upon interaction with PAs. Figure 6d–f showed the amide I (1700–1600 cm⁻¹) and amide II (1575–1480 cm⁻¹) regions of native HEWL and HEWL + PA complexes. Both peaks shifted upon binding to PAs. Amide I moved from 1659 (native HEWL) to 1654 cm⁻¹ (HEWL + FA), 1655 cm⁻¹ (HEWL + CA) and 1654 cm⁻¹ (HEWL + CGA). Amide II shifted from 1546 to 1544 cm⁻¹, 1544 cm⁻¹ and 1547 cm⁻¹ in the presence of FA, CA and CGA, respectively, indicating complex formation.

Amide I was further analysed through deconvolution to estimate secondary structural components. From Figure S15, the α‐helical content of HEWL decreased from 28.56% to 24.02%, 23.34% and 22.46% upon binding FA, CA and CGA, respectively, while other structures increased, indicating structural rearrangement of the protein.

The FT‐IR region of 1500–1100 cm⁻¹, associated with CH₂ and CH₃ bending vibrations of hydrophobic residues, also showed peak shifts upon complexation, suggesting the involvement of hydrophobic interactions.

2.9. Thermal Analysis

Steady‐state fluorescence investigated the thermal stability of HEWL in the absence and presence of PAs. The fluorescence emission of Trp residues was monitored from 298 to 363 K, and refolding was assessed by cooling the system stepwise back to 298 K. Figure S16 showed the fluorescence spectra of free HEWL and HEWL + PA complexes during unfolding and refolding.

Native HEWL exhibited a 9 nm redshift when the temperature increased from 298 to 363 K, indicating protein unfolding. Upon cooling back to 298 K, the emission maximum shifted to 348 nm but with lower fluorescence intensity compared to the initial state, suggesting incomplete refolding. When HEWL was complexed with FA/CA/CGA at 298 K, the emission maxima appeared at 344/347/350 nm, respectively. Upon heating to 363 K, these shifted to 356 nm (HEWL + FA), 354 nm (HEWL + CA) and 356 nm (HEWL + CGA), indicating similar unfolding behaviour for native and complexed HEWL. Analysis of the reversibility spectra suggested that PAs did not significantly enhance refolding of thermally denatured HEWL. From plots of emission maxima versus temperature (Figure S17), native HEWL showed a T _m of 68.6 °C, which increased to 69.6 and 74.0 °C in the presence of FA and CA, respectively, indicating improved thermal stability. However, CGA reduced the T _m to 67.1 °C, suggesting a slight loss of conformational stability. This decrease cannot be fully attributed to the reduction in α‐helical content observed upon CGA binding [53].

2.10. Enzyme Activity

Lysozyme activity can be enhanced or inhibited by small ligand modulators, and ligand‐induced secondary structural changes may alter enzyme function. CD and FT‐IR results indicated structural changes in HEWL upon PA binding, prompting evaluation of its lytic activity against M. lytus in the absence and presence of them. As shown in Figure S18, HEWL activity decreased in the presence of PAs, indicating that these compounds act as enzyme inhibitors. This inhibition may arise from PAs binding near the catalytic residue Asp52, which is essential for HEWL activity. Similar inhibition has been reported [54]. Additionally, Glu35, another key catalytic residue, exhibited increased root mean square deviation (RMSD) values upon PA binding (Section 2.11.2), suggesting its involvement in the interaction. These findings indicate that PA binding induces conformational changes that affect the catalytic function of HEWL.

2.11. Computational Techniques

2.11.1. Molecular Docking

Molecular docking generated nine conformations for each HEWL + PA complex, and the pose with the lowest binding energy was selected for analysis. Figure 7a,d,g displayed the lowest‐energy docked conformer of HEWL with PAs, along with the corresponding 2D interaction plots. The binding energies of all docked poses are listed in Table S7.

FA formed an H‐bond with Asn103, while van der Waals interactions occurred with Asp52, Gln57, Ile58, Asn59, Trp62 and Trp63. π–alkyl interactions were observed with Ile98 and Ala107, and a π–π T–shaped interaction with Trp108 (Figure 7b). CA formed single H‐bonds with Asn103 and Trp108 and two H‐bonds with Gln57. van der Waals interactions with Asp52, Leu56, Ile58, Asn59 and Asp101 were also observed. π–alkyl interactions involved Ala107 and Ile98, while Trp63 formed a π–π T–shaped interaction (Figure 7e). CGA formed H‐bonds with Gln57, Trp62 and Trp 63 and van der Waals interactions with several residues, including Glu35, Asp52, Ile58, Asn59, Ile98, Asp101, Gly102, Asn103, Gly104, Ala107, Trp108 and Val109 (Figure 7h). These residues involved in complexation with PAs have been observed in other literature [44, 46]. The distance for each interaction has been tabulated in Table 3.

TABLE 3.

Changes in accessible area and distances of catalytic residues of HEWL near FA, CA and CGA.

Residues	HEWL + FA		HEWL + CA		HEWL + CGA
	Distance, Å	ΔASA, Å2	Distance, Å	ΔASA, Å2	Distance, Å	ΔASA, Å2
Glu35	—	—	—	—	4.06 (7‐C=O)	5.67
Asp52	4.15 (4‐OH)	18.01	4.58 (4‐OH)	18.93	3.67 (7‐OH)	20.93
Leu56	5.23 (4‐OH)	1.24	5.04 (3‐OH)	1.24	—	—
Gln57	2.72 (4‐OH)	4.49	1.96 (4‐OH), 2.11 (3‐OH)	4.49	2.73 (3‐OH)	3.12
Ile58	3.93 (5‐C)	3.24	3.67 (3‐OH)	2.27	2.87 (3‐OH)	1.37
Asn59	3.89 (3‐OCH3)	29.14	3.92 (5‐C)	19.89	1.94 (3‐OH)	25.58
Trp62	4.51 (3‐OCH3)	18.50	5.41 (6‐C)	17.82	2.88 (9′‐C=O)	19.29
Trp63	3.44 (7‐C)	34.07	3.32 (Cg)	28.8	2.25 (4‐OH)	36.46
Ile98	2.46 (Cg)	2.54	4.48 (Cg)	1.21	3.74 (7′‐C)	4.29
Asp101	—	—	4.27 (4‐OH)	8.11	4.15 (3′‐OH)	25.24
Gly102	—	—	—	—	3.75 (4′‐OH)	3.60
Asn103	2.40 (9‐C=O)	23.37	2.48 (9‐C=O)	26.59	1.90 (3′‐OH)	26.28
Gly104	—	—	—	—	3.73 (9′‐C=O)	13.39
Ala107	3.99 (Cg)	12.10	3.80 (Cg)	14.32	3.15 (4‐C)	9.61
Trp108	4.28 (Cg)	16.76	2.88 (3‐OH)	14.17	3.48 (1‐OH)	18.74
Val109	—	—	—	—	3.97 (7‐C=O)	26.21

The calculated accessible surface area (ASA) values decreased from 6613.12 Ų for native HEWL to 6515.93, 6524.92 and 6456.22 Ų upon binding FA, CA and CGA, respectively. Significant ASA changes (>10 Ų) for Trp62, Trp63, Trp108 and other residues further indicated their involvement in interacting with PAs (Table 3). Surface hydrophobicity analysis (Figure 7c,f,i) showed that the PAs occupied a hydrophobic region of HEWL, indicating a major contribution from hydrophobic interactions.

2.11.2. MD Simulation

To investigate conformational fluctuations in HEWL + PA complexes, MD simulations of all were analysed over 100 ns. Structural snapshots at 0, 50 and 100 ns (Figure S19) showed the PAs largely retained similar positions within the binding pocket, with only minor changes in the overall protein structure.

RMSD analysis (Figure 8a) revealed that HEWL + CA remained the most stable complex throughout the simulation, exhibiting minimal structural deviation. In contrast, HEWL + FA and HEWL + CGA both showed increased RMSD (∼0.08 nm) from 65 and 51 ns, respectively, similar to free HEWL with an RMSD increase of about ∼0.1 nm from 92 ns onwards. HEWL + CGA stabilised after a conformational shift at ∼52 ns, whereas HEWL + FA exhibited an even larger deviation, reaching ∼0.34 nm near 90 ns (Figure 8a). These findings suggested CA maintains protein structure stability, whereas FA and CGA allow for larger changes. Root mean square fluctuation (RMSF) (Figure 8b) revealed that while all four systems maintained broadly similar fluctuation patterns and peaks, PA‐induced differences were evident in peaks spanning the loop region, residues 15–23, 45–48, 61–71, 103–105 and the C‐terminal stretch. Notably, HEWL + CGA and HEWL + FA showed larger peaks in these areas as compared to HEWL + CA, with a more pronounced increase from residue 109 towards the C‐terminal. These residue‐level fluctuations may partially account for the observed RMSD differences. Further supporting these observations, radius of gyration (R _g) analysis (Figure 8c) showed that free HEWL and HEWL + CA both maintained a consistently stable R _g (∼1.39–1.42 nm), reflecting preservation of native‐like compactness throughout the trajectory. In contrast, HEWL + CGA exhibited a structural transition, where an increase in R _g peaking at ∼1.45 nm coincided with the increase in RMSD (∼0.08 nm) around ∼51 ns, indicating structural expansion driven by elevated RMSF amplitudes across key loop regions on CGA binding (Figure 8c). Conversely, HEWL + FA underwent transient compaction (∼24–58 ns; R _g ∼1.37–1.39 nm) followed by re‐expansion (Figure 8c), likely contributing to the two RMSD transitions and indicating compaction‐driven conformational rearrangement. Collectively, the convergence of RMSD, RMSF and R _g analyses indicated that CA most effectively preserves the native structural integrity of HEWL, whereas FA and CGA induce distinct ligand‐specific conformational dynamics. Analysis of cleft residues revealed that, despite similar RMSD values (Table 4) indicating preserved binding site geometry, RMSF differences revealed ligand‐specific modulation of residue dynamics, with Trp62, Asp101 and Gly102 showing significantly reduced fluctuations, and Glu35, Gln57, Gly104 and Val109 exhibiting increased flexibility.

FIGURE 8.

(a) RMSD, (b) RMSF and (c) R _g plots of HEWL, HEWL + FA, HEWL + CA and HEWL + CGA.

TABLE 4.

Average RMSD and RMSF values of the residues that are available in the cleft region of HEWL in its free and bound conformers.

Residue	RMSD, nm				RMSF, nm
	HEWL	HEWL + FA	HEWL + CA	HEWL + CGA	HEWL	HEWL + FA	HEWL + CA	HEWL + CGA
Glu35	0.08	0.11	0.10	0.10	0.10	0.14	0.20	0.14
Asp52	0.07	0.06	0.07	0.07	0.08	0.09	0.08	0.10
Leu56	0.12	0.12	0.12	0.12	0.08	0.09	0.09	0.08
Gln57	0.10	0.10	0.10	0.09	0.01	0.11	0.11	0.10
Ile58	0.03	0.02	0.03	0.03	0.06	0.06	0.06	0.06
Asn59	0.02	0.01	0.02	0.04	0.06	0.06	0.05	0.09
Trp62	0.06	0.05	0.05	0.04	0.15	0.16	0.11	0.11
Trp63	0.03	0.05	0.04	0.04	0.10	0.08	0.08	0.08
Ile98	0.02	0.03	0.02	0.03	0.07	0.08	0.07	0.08
Asp101	0.10	0.12	0.11	0.12	0.27	0.13	0.16	0.11
Gly102	0.03	0.02	0.02	0.02	0.23	0.15	0.16	0.13
Asn103	0.09	0.08	0.08	0.09	0.23	0.23	0.21	0.21
Gly104	0.01	0.03	0.05	0.03	0.11	0.20	0.13	0.20
Ala107	0.01	0.01	0.01	0.01	0.13	0.11	0.13	0.12
Trp108	0.02	0.08	0.06	0.07	0.11	0.12	0.11	0.13
Val109	0.11	0.11	0.11	0.10	0.12	0.20	0.21	0.15

HEWL + PA interactions at this cleft region were analysed during the simulations (Figure S20). The results (Table S8) indicated H‐bonds were the predominant interactions throughout the simulation, followed by hydrophobic π–alkyl interactions. Residues involved in H‐bond includes Asn46, Asp52, Gln57, Asn59, Trp63 and Asn103. HEWL + CGA exhibited the highest H‐bond count (8–9), driven by additional contributions from Trp62 and Asp101, whereas HEWL + CA remained stable (5–7) and HEWL + FA showed a gradual increase (3–6) over time. π–π T‐shaped interactions involved Trp62 and Trp108, while Ile98, Ala107 and Val109 participated in π–alkyl interactions. Hydrophobic interactions were most diversified in HEWL + CGA, whereas HEWL + FA shifted from π–π T‐shaped to π–π stacked, π–alkyl, and alkyl contacts over time, while HEWL + CA showed relatively stable π–π T‐shaped and π–alkyl interactions.

This interaction behaviour is consistent with Molecular Mechanics Poisson‐Boltzmann Surface Area (MMBPSA) analysis of the last 20 ns of the simulation trajectory (Table 5), where HEWL + CGA shows the most favourable binding free energy, followed by HEWL + CA and HEWL + FA (Table 5). The higher H‐bond occupancy in HEWL + CGA aligns with its strongly favourable electrostatic contribution, with lower contributions observed in HEWL + CA and HEWL + FA. CGA binds strongly to HEWL, driven by the highest contributions from E _ele and ΔG _PB. A similar trend observed for HEWL + FA/CA indicates electrostatic and polar interactions dominate complex formation with lesser influence from E _vdw and ΔG _SA. Overall, CGA provided the strongest binding, while CA ensured greater conformational stability.

TABLE 5.

MMBPSA MD simulation calculated energy components.

System	Energy, kJ mol−1
	Total binding (a + b + c + d), kJ mol−1	van der Waals (a), kJ mol−1	Electrostatic (b), kJ mol−1	Polar solvation (c), kJ mol−1	SASA (d), kJ mol−1
HEWL + FA	−118.27 ± 15.87	−98.82 ± 14.12	−114.67 ± 25.31	107.53 ± 12.86	−12.30 ± 1.01
HEWL + CA	−201.27 ± 18.64	−62.53 ± 13.73	−267.17 ± 28.08	139.70 ± 13.46	−11.26 ± 0.63
HEWL + CGA	−273.66 ± 40.93	−82.34 ± 23.35	−440.68 ± 75.50	266.37 ± 23.61	−17.01 ± 0.84

2.12. Reasoning Into the Aggregation Inhibiting Properties of PAs

The results demonstrated the aggregation disruption properties of PAs, indicating their ability to preserve HEWL structural integrity. Among the compounds, CA exhibited the strongest inhibitory activity, followed by FA and CGA. This trend correlates with the binding constants obtained, where HEWL + CA showed the highest affinity, followed by HEWL + FA and HEWL + CGA. Previous studies have reported that stronger protein–ligand binding often results in enhanced stabilisation of the native protein structure and improved fibril inhibition [55]. MD simulations further supported this observation, indicating that CA provides greater structural stability to HEWL throughout the simulation period compared to FA and CGA. Similar correlations between binding affinity and fibril inhibition have also been reported for other protein‐ligand systems [55].

Structural features of these PAs, including the conjugated C=C bond and carboxyl group, provide rigidity that may disrupt intermolecular H‐bonding involved in aggregate formation [56, 57]. Furthermore, the aggregation‐inhibiting activity of the PAs observed here is also influenced by the number and position of hydroxyl groups on the phenolic ring, with consecutive hydroxyl groups enhancing inhibitory effects [58, 60]. CA contains two adjacent hydroxyl groups at the C‐3 and C‐4 positions, whereas FA contains only one, which likely contributed to the stronger inhibitory activity of CA. Although CGA contains several hydroxyl groups, the bulky quinic moiety may have hindered its penetration into the HEWL binding pocket, reducing its inhibitory potential.

Furthermore, the amide pi‐stacking effect during HEWL + FA/CA interaction could have introduced steric hindrance, impacting the distribution of internal hydrophobic residues within the core of HEWL and impeding further aggregation [61]. Several HEWL regions (residues 49–64 and 57–107) are known to be susceptible to amyloidosis [62]. Sugimoto et al. further identified residues 54–62 (K‐peptide region), including Trp62, as crucial for fibrillation [63]. Docking results showed CA/FA interacted with these residues in this aggregation‐prone region, thereby reducing aggregation, whereas CGA interacted with fewer residues, explaining its weaker inhibition (Figure 9). The interaction within the K‐peptide region suggested that binding at these sites may interfere with early nucleation events. This is evident from the FESEM images, which showed the disruption of aggregated HEWL in the presence of PAs.

FIGURE 9.

Surface depiction of the binding site of HEWL for (a) FA, (b) CA and (c) CGA along with the K‐peptide region. Red: FA/CA/CGA, yellow: residues belonging to the K‐peptide region, orange: Trp62 and green: other residues susceptible to amyloid aggregation.

Several natural polyphenols have previously been reported to inhibit HEWL fibrillation. Curcumin suppresses amyloid formation through direct binding to HEWL, stabilisation of native‐like conformations and disruption of β‐sheet assembly [64]. Similarly, EGCG and ECG inhibit fibrillation through aromatic and hydrophobic interactions with aggregation‐prone regions of the protein [65]. Myricetin also effectively prevents HEWL fibrillogenesis by binding to aggregation‐prone regions and reducing the population of partially unfolded intermediates acting as precursors for amyloid formation [66]. Taking into consideration the different aggregation methodologies followed by us and reported literature, our study showed that the tested PAs exhibited aggregation inhibiting activity, as fructose could only induce aggregation and not fibrillation. However, unlike curcumin, catechins and myricetin, which possess extended polyphenolic scaffolds and multiple aromatic rings, FA, CA and CGA are structurally simpler. Despite their smaller molecular size, they were still capable of modulating HEWL conformation and disrupting aggregation. Furthermore, our study establishes a structure–activity relationship among the PAs and correlates their binding characteristics, thermodynamic parameters and MD data with their aggregation inhibiting efficacy, providing mechanistic insights into how structural differences among PAs influence aggregate inhibition.

3. Conclusion

In summary, the PAs under investigation exhibited significant aggregation‐disrupting properties in a time‐dependent manner. Despite the relatively small difference in inhibition between the two concentrations used, this may be attributed to (1) partial saturation of HEWL + PA interactions within the tested concentration range, (2) a non‐linear relationship between inhibitor concentration and aggregation suppression in this system or (3) exploring higher concentrations to achieve a more pronounced inhibitory effect. Nonetheless, the findings provide clear evidence that these compounds effectively inhibit HEWL aggregation under the studied conditions. Among the compounds, CA showed the strongest aggregate inhibition. These observations were linked to interaction studies between HEWL and the PAs. The intrinsic fluorescence of HEWL was quenched in the presence of PAs, while the binding constants indicated moderate interaction. Thermodynamic analysis suggested that hydrophobic interactions mainly governed complex formation. Microenvironmental changes around Trp residues were noted due to HEWL and PA interaction, while CD and FT‐IR showed reduced α‐helical content upon PA binding. MD simulations showed that HEWL remained structurally stable when bound to CA compared with FA/CGA. Docking revealed that FA and CA interact with residues within the K‐peptide region associated with amyloid formation, explaining their stronger inhibitory effects compared with CGA. Overall, these findings provide a basis for further in vivo investigation of PAs as potential‐aggregate inhibiting agents.

4. Experimental Section

4.1. Materials

HEWL (98% purity; L6876) and Micrococcus lysodeikticus (ATCC 4698; M3770) were purchased from Sigma–Aldrich Chemical Co. FA (H0267), CA (C0002) and CGA (C0181) were obtained from TCI chemicals. Monobasic and dibasic phosphate salts used to prepare 20 mM phosphate buffer (pH 7.4) were purchased from SRL. A HEWL stock solution was prepared in this buffer [67] and its concentration determined spectrophotometrically (ε ₂₈₀ = 37,646 M⁻¹ cm⁻¹). PAs were dissolved in absolute ethanol due to low aqueous solubility and further diluted so that the final ethanol concentration remained below 3%. ThT (RM1036), anilino‐1‐naphthalene‐sulphonic acid (1002295874) and CR (00884400025) were procured from Pallav Chemicals, Sigma–Aldrich and HiMedia, respectively.

4.2. Methodologies

A detailed description of the experimental addressed in the supplementary information. The aggregation inhibition properties of two concentrations (100 and 200 μM) of PAs against glycation‐induced aggregation of HEWL has been elaborated in the supplementary information, including FESEM and far‐UV CD studies. UV–vis spectroscopy was executed to determine ground‐state complexation between 20 µM HEWL and 20 µM PAs in the 200–700 nm wavelength region. Steady‐state fluorescence was carried out between 3 µM HEWL in the presence of increasing concentration of PAs (0–16.4 µM) at three temperatures, 293, 300 and 307 K, using an excitation wavelength of 295 nm and recording the emission spectra at 305–500 nm. A slit width of 5/5 nm was maintained in all studies. Prior to the addition of these compounds, the protein was equilibrated for 10 min at each temperature. An average of three titrations was used to determine various parameters established through steady‐state fluorescence. Thermal analysis was also conducted for free HEWL and HEWL‐bound PAs to determine the melting temperature (T _m) of HEWL. This was carried out by examining the fluorescence emission maxima of Trp residue (λ _ex = 295 nm) in the temperature range of 298–363 K. Microenvironmental changes in the protein structure upon binding to each PA was observed using synchronous, 3D fluorescence and REES. CD and FT‐IR was used to examine secondary structural changes of HEWL upon interacting with each PA. The far‐UV CD spectra of 20 µM HEWL and HEWL + PAs complexes (1:2 and 1:5 molar ratios) were recorded in the wavelength range of 190–240 nm. FT‐IR was performed on 10 mg mL⁻¹ of native HEWL and a 1:2 molar ratio of HEWL‐bound PAs using a 256 scan interferogram. The enzyme activity of 10 µM HEWL and HEWL complexed with PAs (1:5 and 1:10 molar ratio) was tested against the clearance ability of the protein against Micrococcus lysodeikticus. AutoDock vina was employed to dock HEWL with each PA, whereby each docked pose yielded binding energies. The docked pose with the lowest binding energy was subsequently used for further analysis. The lowest binding energy docked conformers of HEWL and PAs, and unbound HEWL (control), were subjected to MD simulation using the Groningen Machine for Chemical Simulations (GROMACS) package (version 2020.4), parameterised with GROMOS54a7 molecular force field. MMPBSA was employed to calculate binding energies.

Author Contributions

Sona Lyndem: investigation, methodology, writing – original draft, writing – review & editing, data curation. Sibasree Hojaisa: data curation, methodology. Roohi Choudhury: investigation, methodology, writing – original draft. Sadia Nudrat: methodology, software. Kalpana Kumari: methodology, investigation. Anupam Nath Jha: investigation, methodology, writing – original draft, writing – review & editing. Atanu Singha Roy: writing – review and editing, writing – original draft, conceptualisation, methodology, investigation, supervision.

Funding

This study was supported by Council of Scientific and Industrial Research, India (01/2903/17/ EMR‐II).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supplementary Material

Lyndem Sona, Hojaisa Sibasree, Choudhury Roohi, Nudrat Sadia, Kumari Kalpana, Jha Anupam Nath, Singha Roy Atanu, Complexation of Hen Egg White Lysozyme With Bioactive Phenolic Acids: Molecular Insights Into the Binding Mechanisms and Inhibitory Effects Against Protein Aggregation, ChemPhysChem 2026, 27, e70500. 10.1002/cphc.70500

Data Availability Statement

The data that supports the findings of this study are available in the supplementary material of this article.