Pb²⁺ binding to lentil lectin and its influence on the protein aggregation

Abstract

Binding of Pb²⁺ to lentil lectin (LL) was studied by spectroscopy, microscopy and thermodynamics and the species were modelled by molecular dynamics (MD). The effect of pH on Pb²⁺ binding was studied at pH = 5 in acetate buffer and pH = 7.2 in Tris buffer. Based on ITC, multiple binding sites were found for Pb²⁺ at pH = 7.2. No binding is noticed at pH = 5. The MD results showed the involvement of aspartate and glutamate with hemi-directed geometry for Pb²⁺. Pb²⁺ mediated β sheet to α-helix transition was noticed at pH = 7.2. At physiological pH, morphological changes were observed in the reduction of size of the particles of LL (160 ± 30 nm) to those in {LL + Pb²⁺} (45 ± 10 nm) as derived based on AFM and TEM. In presence of Pb²⁺, the larger particles break down to smaller ones because of the coordination ability of Pb²⁺ towards carboxylate and imidazole moieties. At pH = 5, the TEM shows much higher aggregation than that observed at pH = 7.2 for the protein alone, leading to linear aggregated species which were further broken down by Pb²⁺ into smaller ones.

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Introduction

Heavy metals, such as lead and mercury pose a major threat to human health owing to their interaction with proteins. Physiological effects of Pb²⁺ include neurological disorders, anaemia, kidney damage, hypertension and dampening of male fertility.^1–3 Pb²⁺ can replace both Ca²⁺ and Zn²⁺ in several proteins.^4,5 In humans, the intracellular availability of Pb²⁺ in major organs like the kidney and brain depends upon its complexation with proteins rich in aspartate and glutamate.⁶ In the case of toxic metal-sensing proteins, it is understood that Pb²⁺ interacts and binds to carboxyl, imidazole and cysteine-rich sites.^7–9

Binding of human ​α-lactalbumin with Pb²⁺ has been studied only by fluorescence and it was noticed that the binding of lead decreases the thermal stability of the protein, however, no structural or aggregational information is known.¹⁰ Even the interaction of human serum albumin with Pb²⁺ results in the agglomeration of the protein that is being studied by fluorescence and Raman spectroscopy.³ Even though the agglomeration is noticed in this case, there have been no studies of microscopy in the literature to support the structural features of the aggregates. The literature studies are devoid of ITC data that pertain to the thermodynamic details of Pb²⁺ to these proteins. All these observations support that the proteins and inorganic species deserve thermodynamic, spectroscopy and microscopy studies to reveal the aspects relevant to binding and to the aggregation of the protein in the presence of the added metal ion, and in the present case, Pb²⁺.

Plant proteins, such as, Nicotiana tabacum calmodulin-binding protein (NtCBP4) and Arabidopsis thaliana acyl-CoA-binding protein (ACBP1), are known to bind with Pb²⁺ and thus help to increase the tolerance of the plant from Pb²⁺ toxicity.^11,12 Plant lectins are also metalloproteins which have been widely studied for the metal binding studies.^13,14 Lectins have been extensively used as tools in many areas of biological research due to their carbohydrate binding abilities.^15,16 Since lectins are known to be targeted by heavy metal ions, probing their interactions with such proteins are of immense importance to the understanding of their chemico-biological aspects. The crystal structure of human protein galectin 1 shows striking resemblance to the dimer of lentil lectin (LL dimer).¹⁷ Because of this striking similarity in the quaternary structure of both these proteins, the Pb²⁺ studies were carried out using lentil lectin (LL).

To our knowledge we have not come across any studies of binding of Pb²⁺ to lentil lectin. However, binding of Zn²⁺, Cd²⁺ and Co²⁺ to lentil lectin (LL) was reported.¹⁸ Therefore, this paper is focused at the studies of the binding of Pb²⁺ to LL by spectroscopy and thermodynamics, and the protein aggregation by microscopy. The binding of Pb²⁺ to LL was also modeled by molecular dynamics study.

Experimental

The protein, LL has been isolated, purified and characterized as per the literature reported method.¹⁹ The crude LL was further purified over mannose affinity chromatography followed by dialysis against 10 mM Tris buffer, pH = 7.2 and was checked by SDS-PAGE before assaying by haemagglutination (Fig. SI–01†) and found that the protein is pure. Protein concentration was determined spectrophotometrically using an extinction coefficient of 3 × 10⁴ M⁻¹ cm⁻¹ at 280 nm.²⁰

Fluorescence, absorption and CD titrations

Fluorescence studies were performed on Perkin Elmer LS 55 Fluorescence Spectrometer. A 20 μM of LL in 5 mM Tris buffer at pH = 7.2 was used and a 100 μL of this solution was made up to 1 mL using the buffer. The Pb²⁺ titrations of LL were carried out by continuous addition of this ion starting from 1 to 10 μL of a 100 mM of stock solution of lead perchlorate to 1 mL of 2 μM protein solution (in cuvette). In cuvette, the Pb²⁺ concentration comes to 0.1 to 1 mM. Such concentrations of Pb²⁺ were indeed used in the literature for the titration of human α-lactalbumin.¹⁰ Similar titrations were carried out in UV-visible absorption studies on Varian Cary 100 UV-Spectrophotometer. A control was performed by titrating the buffer similarly and subtracting the same from the protein data obtained in the corresponding titration. Far- and near-UV CD spectra were measured using a Jasco J-815 CD spectrometer using a 0.1 cm cuvette. Protein samples were studied at a concentration of 4 μM and 50 μM for far- and near-UV CD respectively.

FTIR and NMR spectra

The FTIR spectra were measured on a Bruker tensor-27 spectrometer. The resolution was set at 2 cm⁻¹ and the path length was 0.075 cm. Total scans were 400 for all the sample and the background. ²⁰⁷Pb NMR spectra were collected using a Bruker 400 spectrometer equipped with a 5 mm broad-band probe. The ²⁰⁷Pb chemical shift was externally calibrated using 1.0 M Pb(NO₃)₂ in D₂O solution, resonating at −2861.2 ppm.²¹

Isothermal titration calorimetry (ITC)

The calorimetric titrations were performed using isothermal titration calorimeter from MicroCal (Northampton, MA, USA). A 5 mM Pb²⁺ taken in syringe and LL (10–60 μM) in the calorimeter cell. Successive additions of 1 μL of Pb²⁺ were separated by a 150–200 s interval to allow the exothermic/endothermic peak resulting from the [LL + Pb²⁺] interaction to return to the baseline. The ITC data were fitted with the origin software package provided by MicroCal.

Sample preparation for AFM and TEM

In order to carry out the microscopy studies, 60 μM (250 μl) of protein was incubated with 5 mM (40 μl) of Pb²⁺. The sample was then sonicated for 10 min. For AFM, the sample was spread over mica sheet by drop cast method, dried and analyzed by multimode Veeco Dimensions 3100 SPM with Nanoscope IV controller instrument. Similarly, even for the TEM studies, the samples were spread over carbon coated copper grids having 200 mesh and the sample was dried and analyzed by PHILIPS CM 200 transmission electron microscope operating at 120 kV.

Molecular dynamics (MD) simulations

Since, the simulation of the LL dimer demands large computational times, and further there exists a striking resemblance between the two subunits of LL, only one monomer was considered for MD simulations. These were performed with explicit solvent condition under periodic boundary conditions using GROMACS 4.5.1 software with amber03 force field.^22,23 Several Pb²⁺ ions were placed around the protein randomly using accelrys DS visualizer before subjecting to the simulation study. The solvation was done with 22 000 TIP3P water and the charge balancing (Cl⁻) were done using routine procedures. This was then energy minimized for 20 000 steps with SD method²⁴ followed by position restraint for 100 ps. Finally the entire system was allowed to go for 70 ns simulation under NTP condition with 2 fs time step. Protein, Pb²⁺, Cl⁻ and the solvent were coupled separately to V-rescale temperature bath. Parrinello–Rahman bath was used for pressure coupling. The electrostatic interactions were calculated by using the particle mesh Ewald summation method.²⁵ All the bond lengths were constrained with LINCS.²⁶

Results and discussion

Emission

The fluorescence studies (Fig. 1) carried out with Pb²⁺ in 20 mM acetate buffer at pH = 5 showed only marginal quenching in the intensity of 335 nm band of LL when excited at 280 nm. On the other hand, the studies in 5 mM Tris buffer at pH = 7.2 exhibited significant fluorescence quenching, suggesting that the binding of Pb²⁺ is more at pH = 7.2 when compared to that at pH = 5. This has been further confirmed based on ITC studies.

Fig. 1 Fluorescence titration of [LL] = 2 μM upon addition of [Pb²⁺] = 0.1–1.5 mM: (a) in 20 mM acetate buffer, pH = 5; (b) in 5 mM Tris, 10 mM NaCl, and pH = 7.2; (c) I/I₀ vs. Pb²⁺ concentration plot in acetate (■) and in Tris buffer (●).

Absorption

The absorption spectra measured with Pb²⁺ in 20 mM acetate buffer at pH = 5 (Fig. 2a and b) exhibited no significant change in the absorbance of 280 nm band. However, major changes were observed in the absorbance at 218 nm band and the intensity gradually decreases with the addition of Pb²⁺ owing to a decrease in protein π⋯π interactions.²⁷

Fig. 2 (a) Absorption spectra of [LL] = 15 μM upon addition of [Pb²⁺] = 0.2–1.5 mM in 20 mM acetate buffer (pH = 5), (b) absorbance vs. Pb²⁺ concentration plots at 218 (■) and 280 (●) nm, (c) absorption spectra of [LL] = 15 μM upon addition of [Pb²⁺] = 0.2–1.5 mM in 5 mM Tris, 10 mM NaCl, and pH = 7.2 expanded spectra (200–300 nm) is given in inset. (d) Absorbance vs. Pb²⁺ concentration plots at 235 (■), 207 (●), and 280 nm (▲).

The absorption spectra measured with Pb²⁺ in 5 mM Tris buffer at pH = 7.2 (Fig. 3c and d) showed increased absorbance for 280 nm band suggesting that the protein is susceptible for Pb²⁺ binding. In addition, a new absorption band appears at 220 nm. The decrease in the absorbance of ∼210 nm band with the addition of Pb²⁺ suggests the interaction of this ion even with the peptide back bone region. At pH = 7.2 the de-protonated histidines interact with Pb²⁺ while the same is not true at pH = 5 owing to the protonation. The sharp contrast observed between the absorption spectra at pH = 5 and 7.2 is attributable to the availability of only the –COOH groups at pH = 5, while carboxylate and imidazolate groups at pH = 7.2.^28,29 The new band at 240 nm is due to the histidine-N → Pb²⁺ and tyrosine-O⁻ → Pb²⁺ charge transfer transitions. This is supported by the literature data.^28,29

Fig. 3 (a) Far-UV CD spectra of [LL] = 4 μM upon addition of [Pb²⁺] = 0.1(red) − 1(pink) mM in 5 mM Tris, 10 mM NaCl and pH = 7.2. (b) Near-UV CD spectra of [LL] = 10 μM with [Pb²⁺] = 1–5 mM in the Tris buffer. (c) Far-UV CD spectra of [LL] = 4 μM titrated against [Pb²⁺] = 0.1–1 mM in 20 mM acetate buffer at pH = 5.

The binding of Zn²⁺ to metallothionein results in the formation of new absorption band at 240–250 nm and this was attributed to the interaction of metal ions with protein as reported in the literature.^30,31 In our work, the aromatic band at ∼220 nm splits, diminishes and forms a new band as a function of addition of Pb²⁺ concentration and the new band is observed at 240 to 250 nm. Thus the absorption spectral changes observed in our case with LL protein closely matches with that observed for the metallothionein–metal ion interaction (Fig. 2b {inset}).

Circular dichroism

CD spectra were measured for the titration of LL by Pb²⁺ in Tris- as well as in acetate buffers (Fig. 3). The far-UV CD spectrum of LL shows a negative and a positive band at 222 and 197 nm respectively. As LL is titrated with Pb²⁺ at pH 7.2, two new bands start growing as a function of increase in the concentration of Pb²⁺ and these are centered at 213 and 227 nm. The ellipticity of both these bands increases, suggesting an increase in the α-helical³⁰ content in the presence of Pb²⁺. The ellipticity of the band at 197 nm decreases. The secondary structural changes were further supported by FTIR study. However, in the acetate buffer at pH = 5, no significant change was noticed in CD in the far-UV region. At pH = 7.2, near-UV CD spectra exhibit high positive ellipticity for simple LL arising from the aromatic amino acids. This is further supported by a literature report.³¹ The marginal changes observed in CD in presence of Pb²⁺ suggests that no major changes occur in the tertiary structure of LL upon interaction with Pb²⁺.

FTIR

FTIR spectral results were interpreted after subtracting the control spectra (Fig. 4). In LL, only bands corresponding to β-sheet (1636 cm⁻¹) and random coil (1646 cm⁻¹ from) were observed.^32,33 However, in the presence of Pb²⁺ a new band corresponding to α-helix (1657 cm⁻¹) appears suggesting that the Pb²⁺ binding is responsible for the change observed in the secondary structure of LL. On the other hand, no significant change was observed in the FTIR spectra of LL under Pb²⁺ addition in acetate buffer, suggesting no secondary structural change occur in LL in this buffer.

Fig. 4 Fourier – self deconvolved (a and b) and second derivative (c and d) absorption spectra of [LL] = 0.2 mM and [LL + Pb²⁺] respectively, in presence of 5 mM Tris, 10 mM NaCl, pH = 7.2.

²⁰⁷Pb NMR

²⁰⁷Pb NMR spectra were measured to analyze the interaction of LL with Pb²⁺ (Fig. 5). For atoms such as S, O, and N in the biological system, the NMR shielding was found to increase in the order S < N < O.^21,34 An upfield shift of 38 ppm is observed in [LL + Pb²⁺] when compared to simple Pb(ClO₄)₂. In the absence of any cysteine in LL, the NMR shift is attributed to the interaction between Pb²⁺ and carboxylate and imidazole moieties of LL.

Fig. 5 ²⁰⁷Pb NMR spectrum: (a) Pb(NO₃)₂; (b) Pb(ClO₄)₂; (c) 70 μM [LL] was treated with 0.16 M of Pb(ClO₄)₂. Peak (i) and (ii) corresponds to (LL + Pb²⁺), and Pb(ClO₄)₂.

Isothermal titration calorimetry

Isothermal titration calorimetry (ITC) experiments were carried out to explain the thermodynamics of binding of Pb²⁺ to LL at 10, 20, 40 and 60 μM protein concentrations in 5 mM Tris-buffer at pH = 7.2 (Fig. 6). Since considerable heat changes were observed in the titration of the buffer alone by Pb²⁺, the protein titration data were corrected by subtracting the simple buffer titration data and the outcome of this has been used for interpreting the results. While the titrations carried out at 10 and 20 μM of [LL], the titration primarily shows endothermic heat change, those carried out at 40 and 60 μM of [LL] exhibit exothermic followed by endothermic heat changes. No considerable heat changes were observed beyond 50, 30, 10 and 10 {[Pb²⁺]/[LL]} mole ratios, respectively, for 10, 20, 40 and 60 μM of [LL], suggesting the dependence of Pb²⁺ binding on the protein concentration in the ITC cell. In case of 40 and 60 μM of [LL], the transition from exothermic and endothermic occurs around 5 mole equivalents of {[Pb²⁺]/[LL]}. While the ITC data obtained in case of 10 and 20 μM [LL] fits well with one set of binding sites of n = 27 and 17 respectively, those carried out at 40 and 60 μM [LL] fits well with sequential binding sites of n = 7 and 5 respectively. The average binding constants decreases, K (M⁻¹), 3.6 × 10⁵ > 2.0 × 10⁵ > 2.9 × 10⁴ ≥ 2.0 × 10⁴, respectively, as [LL] increases from 10, 20, 40 to 60 μM, suggesting that Pb²⁺ exhibits only a medium strength of binding to the protein. Thus the number of binding sites as well as the strength of binding decreases. The decrease in K is by one order of magnitude on going from low to high [LL] concentration. The binding is associated with positive ΔS in case of 10 and 20 μM [LL], however, at higher [LL] (viz., 40 and 60) some bindings were associated even with negative ΔS. All this agrees well with the fact that an increase in the protein concentration results in higher protein–protein aggregation thus providing less number of sites for metal ion binding.

Fig. 6 ITC of the titration of x μM of [LL] with 5 mM Pb²⁺ in {5 mM Tris, 10 mM NaCl} and pH = 7.2: (a) x = 10; (b) x = 20; (c) x = 40 and (d) x = 60.

In spite of a number of titrations carried out repetitively using both higher and lower concentrations of protein in acetate buffer at pH = 5, no interpretable titration isotherms were observed (Fig. 7) suggesting a stronger aggregation of the protein in this buffer, which was further confirmed based on microscopy studies.

Fig. 7 ITC of the titration of x μM of [LL] with 5 mM Pb²⁺ in 20 mM acetate buffer, pH = 5 (a) x = 10; (b) x = 20; (c) x = 40 and (d) x = 60.

No significant changes were observed in the fluorescence and absorption spectra and no heat changes in the ITC studies in acetate buffer at pH = 5. All this suggests that the Pb²⁺ does not bind either with acetate buffer or with protein (LL). However, in Tris-buffer at pH = 7.2, even after subtracting the controls, the changes were observed in spectra as well as thermodynamics supporting the binding of Pb²⁺ to LL.

Molecular dynamics (MD)

In order to understand the binding features of Pb²⁺ to LL, molecular dynamics (MD) simulations were carried out at 70 ns (Fig. 8) as per the details given in the experimental section. The study resulted in binding of five Pb²⁺ ions to the surface of LL. The Pb1 shows four coordination extended by the carboxylates of Glu 18, Asp 54 and Asp 56 with hemispherical coordination wherein half of the coordination sphere is empty. Such hemi-directed coordination sphere had indeed been reported in the literature in case of Pb²⁺ as well as Ca²⁺ when bound to proteins.^4,5 The remaining four Pb²⁺ ions showed only incipient binding through one carboxylate each of Gly 47 (terminal), Asp 72, Glu 134 and Glu 138 respectively for Pb2, Pb3, Pb4 and Pb5 (Fig. 8) suggesting that the interaction between LL and Pb²⁺ is initiated at the surface of the protein through carboxylate binding, at the 70 ns simulations. The metric parameters of the coordination around Pb1 exhibit a stronger binding through the carboxylates which act as monodentate as compared to that of the bidentate ones. Thus the observed binding pattern indicates that the Pb²⁺ preferably binds to the side chain carboxylates and the binding pattern is similar to that observed in the literature for a hemi-directed binding.^4,5

Fig. 8 From the 70 ns MD simulations of the interaction of Pb²⁺ with LL: (a) Pb²⁺ bound LL; (b) the coordination about Pb1. The metric parameters of the coordination sphere of Pb1 in bond distances (Å) and bond angles (°): O1–Pb1 = 2.256, O2–Pb1 = 2.241, O3–Pb1 = 2.375, O4–Pb1 = 2.334; O1–Pb1–O2 = 81.2, O1–Pb1–O3 = 75.9, O1–Pb1–O4 = 118.7, O2–Pb1–O3 = 93.5, O2–Pb1–O4 = 125.8, O3–Pb1–O4 = 51.9; (c) hemi-directed binding coordination sphere for Pb²⁺ from the literature.^4,5

Microscopy

In order to study the aggregational features of simple protein (LL) and the same in presence of Pb²⁺ under different pH conditions, transmission electron microscopy (TEM) and atomic force microscopy (AFM) experiments were carried out. The TEM micrographs of LL in 5 mM Tris buffer at pH 7.2, as given in Fig. 9a and b, shows well distributed particles over carbon coated copper grid. The micrographs were further analyzed for the size distribution of the particles (Fig. 11a and b). Majority of these particles are elongated with an average size of 160 ± 30 nm. However, in presence of Pb²⁺, the average size of the particle was reduced to 40 ± 10 nm owing to the coordination ability of Pb²⁺ towards the surface exposed carboxylate and imidazole moieties that brings a change in the original aggregation of the protein, as metal ion induced effect (Fig. 10c and d).

Fig. 9 TEM in 5 mM Tris, 10 mM NaCl, pH = 7.2. (a and b) are the micrographs of LL [60 μM] at scale bars 2 and 1 μm respectively. (c and d) are the micrographs of [LL + 5 mM Pb²⁺] at two different regions of the sample.

Fig. 10 Histograms showing the particle distribution from TEM micrographs: (a and b) are for LL and [LL + Pb²⁺] in Tris buffer at pH = 7.2. (c and d) are for LL and [LL + Pb²⁺] in acetate buffer at pH = 5.

On the other hand, in 20 mM acetate buffer at pH = 5, the TEM of simple protein (LL) shows much higher aggregation than that observed in Tris buffer at pH = 7.2, leading to linearly aggregated species (one-dimensional aggregation) with rhombic type structures acting of bricks (Fig. 11a and b) and the particles were analyzed for their sizes (Fig. 10c and d). Such aggregation seems to be a result of the conformational change and also due to the exposed hydrophobic residues of the protein at low pH. The average size of these bricks is (415 ± 125) nm. Upon the addition of Pb²⁺, the aggregated species breaks down to result in smaller particles as can be seen from Fig. 11c and d. The average size of these particles has been dramatically reduced to (38 ± 12) nm owing to the interaction of Pb²⁺ with protein (Fig. 11c and d).

Fig. 11 TEM in 20 mM acetate buffer, pH = 5. (a and b) are the micrographs of LL [60 μM] at scale bars 1 and 2 μm respectively. (c and d) are the micrographs of [LL + 5 mM Pb²⁺] at two different regions of the sample.

In AFM studies over the mica sheet, the LL shows uniformly distributed particles of oval to elongated shape with average size of 135 ± 30 nm in Tris buffer at pH = 7.2 as can be noted from Fig. 12a and e. In presence of Pb²⁺, the larger particles break down into smaller and spherical ones with average size being 40 ± 10 nm (Fig. 12b and f). On the other hand, in the acetate buffer at pH = 5, the LL particles are arranged in a linear fashion and these features are comparable with those observed in TEM for LL at pH = 7.2. The particle distribution is high in density and possesses an average size of 170 ± 45 nm and this is larger than the particles observed at pH 7.2 in Tris buffer (Fig. 12c and g). In the presence of Pb²⁺, the linear array of aggregates further breaks down into smaller particles with average size of 39 ± 12 nm (Fig. 12d and h), a result that was also observed in TEM.

Fig. 12 AFM is in 5 mM Tris, 10 mM NaCl, pH = 7.2. (a and b) are the micrographs of LL and [LL + Pb²⁺]. (e and f) are the corresponding particle size distributions. AFM in 20 mM acetate buffer, pH = 5. (c and d) are the micrographs of LL and [LL + Pb²⁺]. (g and h) are the corresponding particle size distributions.

Conspectus

LL is rich in carboxylate containing amino acids (∼10%), viz., aspartate and glutamate. LL contains three histidines per monomer of which one (His-136) is involved in binding to manganese along with Asp-129 and Glu-119.³⁵ As a result of all these, the binding of Pb²⁺ ions through the available moieties bring appropriate changes in the protein conformation and structure. Histidines provide binding site as supported by the absorption spectra through HisN → Pb²⁺ charge transfer transition as shoulder at 260 nm and the binding of Pb²⁺ was clearly supported by the thermodynamic experiment of ITC study. Indeed the ITC results revealed multiple binding sites. For e.g., at higher protein concentration at pH = 7.2 it shows at least five binding sites. On the other hand, at lower protein concentration, i.e., 10 and 20 μM [LL] more number of binding sites was observed since the protein aggregation is minimal at such concentrations and more surface residues are available for binding. Thus at higher protein concentration, aggregation of the protein dominates. This is understood even from microscopy data.

Further, it should be noted that the aggregation of LL depends on pH and hence at pH = 5 significant aggregation was found as a result of the conformational changes which brings hydrophobic residues more exposed and this drives the aggregation and as a result Pb²⁺ binding is not so much favored at pH = 5.

Fig. 13 shows plausible Pb²⁺ binding core in LL and hemi-directed binding coordination for Pb²⁺ and agrees well with that proposed in the literature.^4,5

Fig. 13 Pb²⁺ binding core in LL with hemi-directed coordination sphere.

The literature studies of Pb²⁺ with other proteins imply binding mainly through carboxylate and histidine, as also shown in the present case. However, in case of Cys containing proteins, the Pb²⁺ additionally binds through cysteine as shown in case of the protein PbrR691.^4,5,36 Thus in case of LL which does not possess any Cys, the binding is only through carboxylates and the histidine, depending upon the pH condition.

Both the CD and FTIR spectra are suggestive of structural changes in LL induced by Pb²⁺. The structural changes that occur in LL upon Pb²⁺ interaction includes increase in α-helical content without affecting the tertiary structure as supported by the CD study. There are hardly any reports on the aggregational behavior of proteins in presence of Pb²⁺ by microscopy. As per a literature report, Pb²⁺ binds to the calcium site in α-lactalbumin where the Ca²⁺ binds through three carboxylate oxygen of Asp82, 87, 88 and two backbone oxygen of Lys79 and Asp84 and two water molecules.¹⁰ The highlights of the results reported in this paper were depicted through Scheme 1.

Scheme 1 Schematic representation of Pb²⁺ binding effect on the aggregational behaviour of LL.

Hence, the present study helps to understand the effect of heavy metal like Pb²⁺ on the structure of proteins which are rich in β-sheet. Since galectin 1, a human lectin has similarity with LL, the Pb²⁺ can interact with galectin¹⁷ in a similar way and may provide some clues regarding the induction of various diseases like cancer and neurogenerative disorders.³⁷

Acknowledgements

CPR acknowledges the financial support from DST (SERB & Nano Mission), CSIR and DAE-BRNS. VKH acknowledge CSIR and KT acknowledge UGC for their fellowships. We thank the central facilities of IIT Bombay for providing us with AFM and TEM data. We thank BRAF (Bioinformatics Resources and Applications Facility) at Centre for Development of Advanced Computing (C-DAC), Pune for computational time.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra06427a

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