Inhibition of amyloid fibrillation of human γD-crystallin by gold nanoparticles: Studies at molecular level

Journal Pre-proof Inhibition of amyloid fibrillation of human γD-crystallin by gold nanoparticles: Studies at molecular level

Vandna Sharma, Shivani Sharma, Shiwani Rana, Kalyan Sundar Ghosh PII:

S1386-1425(20)30177-3

DOI:

https://doi.org/10.1016/j.saa.2020.118199

Reference:

SAA 118199

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date:

24 September 2019

Revised date:

25 February 2020

Accepted date:

25 February 2020

Please cite this article as: V. Sharma, S. Sharma, S. Rana, et al., Inhibition of amyloid fibrillation of human γD-crystallin by gold nanoparticles: Studies at molecular level, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy(2020), https://doi.org/10.1016/j.saa.2020.118199

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© 2020 Published by Elsevier.

Journal Pre-proof Inhibition of amyloid fibrillation of human γD-crystallin by gold nanoparticles: Studies at molecular level Vandna Sharma, Shivani Sharma, Shiwani Rana and Kalyan Sundar Ghosh* Department of Chemistry, National Institute of Technology Hamirpur, Himachal Pradesh

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177005, India

*Corresponding Author

Tel: +91-1972-254104; Fax: +91-1972-223834; e-mail: [email protected]

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Journal Pre-proof Abstract The capability of citrate-stabilized gold nanoparticles (AuNps) has been explored for the inhibition of amyloid fibrillation of human γD-crystallin (HGD), a major protein of eye lens. Citrate-capped AuNps were synthesized, characterized and used further for amyloid inhibition. The results from intrinsic and extrinsic (in the presence of Thioflavin T and ANS) fluorescence based assays and CD spectroscopy clearly suggest that AuNps at nanomolar concentrations can act as an effective inhibitor against fibrillation of HGD. Fluorescence

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microscopic and transmission electron microscopic images also supported this observation.

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Considering the inhibitory role of AuNps against HGD fibrillation, interactions between

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HGD and AuNps were studied to decipher the mechanism of amyloid inhibition. The binding

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and quenching constants were calculated as ~109 M-1 using the data of tryptophan fluorescence quenching of HGD by AuNps. Ground state complexation between the protein

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and nanoparticles was predicted. AuNps were not found to cause any major conformational

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changes in the native protein. Entropy-driven complexation process between the protein and nanoparticles indicates the interactions of AuNps with hydrophobic residues of HGD.

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Therefore, in the presence of AuNps, the exposure of the hydrophobic patches of HGD

Keywords

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during its partial unfolding became restricted, which results inhibition in HGD fibrillation.

Human γD-crystallin; fibrillation inhibition; gold nanoparticles.

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Journal Pre-proof 1 Introduction Cataract, a very common old-age crisis arises due to protein aggregation in eye lens. Lens transparency is meticulously conserved by α, β and γ-crystallin proteins. As a molecular chaperone, alpha-crystallin basically resists the aggregation of beta- and gamma-crystallins. But with the aging of the lens, several factors encourage the progress of crystallins aggregation and these aggregates start to cause light scattering, a hallmark of cataract. Not only the amorphous aggregates, but also some fibrillar and filamentous forms of aggregated

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crystallins were detected in aged and cataractous lens, which also have the potential to scatter

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light [1, 2]. All these three major types of crystallins undergo in vitro amyloid fibrillation

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under the conditions leading to their partial unfolding [3-5]. Amyloid fibrillation of human

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γD-crystallin (HGD), a major γ-crystallin from the nuclear region of lens was studied earlier with detailed structural analysis of fibrils by different research groups [6-8]. The structure of

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HGD is composed of two domains namely N-terminal domain (N-td) and C-terminal domain

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(C-td) [9]. The N-td is less stable as compared to C-td [10]. It was earlier reported that C-td forms the amyloid core and N-td exists in an unordered conformation [7, 8], which suggests

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that the fibrillation of HGD proceeds through its partial unfolding. The deposited amyloids

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can potentially perturb the short-range interactions among various crystallins leading to cataractogenesis. Earlier, L- and D-carnosine were found to prevent amyloid fibrillation of αcrystallin [11]. Our group had also reported the inhibition of HGD fibrillation by using some dye molecules [12]. Inhibitory potential of a green tea polyphenol epigallocatechin gallate against fibrillation of γB-crystallin was reported earlier in this journal [13]. Dissolution of amyloid fibrils of α-crystallin was achieved by using lanosterol [14]. Through screening of several small molecules, an attempt was also made to identify potent pharmacological chaperones to combat fibrillation of α-crystallin [15]. Some peptide molecules had demonstrated inhibition of βB2-crystallin fibrillation through targeting the protein [16].

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Journal Pre-proof In this milieu, it is relevant to note that in last few years, inhibition of amyloid fibrillation of various peptides and proteins was achieved by using different nanomaterials [17]. The nanoparticles coated with hydrophobic molecules and having surface charges were found to be efficient against fibrillogenesis process [18, 19]. In this regard, gold nanoparticles (AuNps) were emerged as compelling inhibitor against fibrillation of Aβ peptide [18-20] and insulin [21]. AuNps capped with citrate can not only inhibit amyloid fibrillation of beta lactoglobulin, but also assist the refolding of the protein into native like conformation [22].

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Heat-induced aggregation of bovine serum albumin, malate dehydrogenase and citrate

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synthase was found to be suppressed by cysteine-capped AuNps through hydrophobic

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interactions between the nanoparticles and hydrophobic regions of the folding intermediates

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[23]. Polyvinylpyrrolidone-conjugated AuNps have also demonstrated inhibitory as well as disaggregation effect against fibrillation of lysozyme [24]. Curcumin-functionalized AuNps

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were able to inhibit and disintegrate the amyloid fibrils of lysozyme and Aβ1-40 [25]. Glycine-

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coated iron oxide magnetic nanoparticles are reported recently for their destroying activity on α-crystallin amyloid fibrils [26]. In general, interactions between the nanoparticles and

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partially folded intermediates of the proteins were depicted as the mechanism of amyloid

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inhibition. In addition to that, it is well-reported in literature that gold nanoparticles bear almost negligible to marginal cytotoxicity and they are non-immunogenic and biocompatible [27-30]. These properties make AuNps very useful for applications in nanomedicine.

Considering the antiamyloid activities of AuNps, we have chosen gold nanoparticles for the inhibition of amyloid fibrillation of HGD in the present work. To best of our knowledge, we are reporting the inhibition of amyloid fibrillation of γ-crystallins by nanoparticles for the first time. In this study, the inhibitory effect of AuNPs against fibrillation of HGD was investigated in vitro using various biophysical assay techniques and microscopic tools.

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Journal Pre-proof Furthermore, the interactions between AuNps and HGD were studied using different spectroscopic techniques to decipher the mechanism of inhibition.

2 Materials and method Thioflavin T (ThT) and 8-anilino-1-naphthalenesulfonic acid (ANS) were acquired from Sigma-Aldrich and tetrachloroauric (III) acid [HAuCl4.3H2O], tri-sodium citrate dehydrate and culture media were purchased from Himedia. UV-Vis absorption and fluorescence

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measurements were carried out using a spectrometer (Lasany make) and a fluorimeter

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(Shimadzu RF-5301PC) respectively. All the fluorescence assays were carried out in

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phosphate buffer (10 mM, pH 7.0). Over-expression and purification of HGD was

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accomplished following the procedure described earlier by Chauhan et al. [31].

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2.1 Preparation and characterization of gold nanoparticles (AuNps)

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AuNps were prepared by reducing chloroauric acid (HAuCl4) using tri-sodium citrate as described by Sharma et al. [32]. Briefly, 10 mL of 1 mM aqueous solution of the gold

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precursor was boiled for 10 minutes. On addition of 1 mL aqueous solution of tri-sodium

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citrate (30 mM) to this boiling solution, a faint blue color was appeared. The whole solution was then boiled for another 10 minutes under continuous stirring and the color of the solution turned wine red. This indicates the formation of gold nanoparticles. Heating was removed and the solution was further stirred for 10 minutes. Finally, a wine red solution of gold nanoparticles was obtained. The hydrodynamic radius and zeta potential of AuNps were determined using Zetasizer instruments (Malvern make). Assuming spherical shape of the nanoparticles and complete reduction of gold (III) to gold atoms, the molar concentration of AuNps was calculated according to the method described earlier [33, 34] (Supplementary materials).

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Journal Pre-proof 2.2 HGD fibrillation HGD (1 mg/mL) was incubated in absence and the presence of different concentrations (0-6.1 nM) of AuNps in 100 mM acetate buffer (pH 3.0) containing 100 mM NaCl at 37˚C for 20 hours. The pH of the incubated mixtures was found to be steady during incubation. To assess the extent of fibrillation, ThT fluorescence assay was used as described below.

2.3 ThT fluorescence assay

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Forty microliters of aliquot from each of the above incubated solutions was added to 50 μM

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of ThT in phosphate buffer. After 10 minutes of incubation, emission spectra (460-600 nm)

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of all these solutions were recorded using an excitation at 442 nm. Each spectrum was

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2.4 Tryptophan (Trp) fluorescence assay

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corrected with respect to their corresponding blank.

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Forty microliters of aliquot from the HGD (1 mg/mL) fibrillar solutions made in the presence and absence of 4 nM of AuNps was mixed with 460 μL of phosphate buffer. Intrinsic

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fluorescence spectra (300-500 nm) of these two solutions were recorded through excitation at

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295 nm.

2.5 ANS binding assay

Forty microliters of aliquot from the HGD (1 mg/mL) fibrillar solutions made in the presence and absence of 4 nM of AuNps was mixed with ANS (20 μM) in phosphate buffer followed by incubation in the dark for one hour. Emission spectra (400-600 nm) of these two solutions were recorded through excitation at 380 nm.

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Journal Pre-proof 2.6 Circular dichroism CD spectra of HGD fibrillar solutions (made in absence and the presence of 4 nM of AuNps) were acquired in the wavelength range of 190-250 nm using JASCO (J-815) spectrophotometer. CD spectra of native HGD with and without AuNps were also recorded. The final protein concentration for far-UV CD measurements was kept at 0.35 mg/mL in 5 mM phosphate buffer (pH 7.0). All the spectra were corrected with respect to their

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corresponding blank.

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2.7 Fluorescence microscopy

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HGD fibrillar solutions were mixed in equal proportion with ThT (50 μM) and were placed

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on a glass slide for drying. Images were captured using Nikon Eclipse TI-U system.

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2.8 High resolution transmission electron microscopy (HRTEM)

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HGD fibrillar solutions were dropped on TEM grids and air-dried. TEM image of only

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AuNps was also captured. TECNAI G2S-Twin TEM was used for imaging.

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2.9 Fluorescence quenching and synchronous fluorscence Emission spectra (300 to 450 nm) of HGD (4 μM) before and after successive addition of AuNps (0-0.55 nM) were recorded by exciting the solutions at 295 nm at four different temperatures (294, 300, 305 and 310K). The recorded emission intensities were corrected for inner filter effect. The fluorescence quenching of the HGD by AuNps was analyzed in terms of Stern−Volmer equation and its modified version [35], which are frequently used for fluorescence quenching studies. The binding constant and the number of binding sites in the protein molecule to interact with AuNps were also determined from the fluorescence data using double

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Journal Pre-proof logarithmic plot [36]. The van’t Hoff equation was used further to calculate the thermodynamic parameters such as change in enthalpy (ΔH), entropy (ΔS) and Gibbs free energy (ΔG) with an assumption that ΔH did not vary significantly over this range of temperatures. Synchronous spectra were recorded after each addition of AuNps (0-0.50 nM) to a solution of HGD (4 μM). The difference between the wavelength of the excitation and emission

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monochromators (Δλ) was set at 60 nm.

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3 Results and Discussion

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3.1 Characterization of prepared AuNps

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The SPR band of AuNps was found to be centered at ~520 nm (Supplementary materials Fig. S1). This resembles with the reported SPR band of AuNps, which also lies in the range of

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520-530 nm [32, 37, 38]. Based on the UV-Vis spectra, it was also found that the synthesized

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AuNPs were quite stable at least for 10 days at 4˚C (Supplementary materials Fig. S1).

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The hydrodynamic radius of the synthesized AuNps was found to be ~15 nm as measured

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using dynamic light scattering (Supplementary materials Fig. S2A). The zeta potential of the nanoparticles was also determined as –44.0 mV (Supplementary materials Fig. S2B). This suggests that the particles were repealing each other and a have low tendency towards agglomeration. The dimension of AuNps was determined from HRTEM image (Fig. 1). In the HRTEM image, the synthesized AuNps were found to be stable at nano size range with an almost spherical shape and a narrow size-distribution. The average diameter of AuNps was 14+0.5 nm. Based on this size, the estimated concentration of the AuNps prepared by us was approximately 12 nM (following the procedure described in Supplementary materials).

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Journal Pre-proof

Fig. 1: HRTEM image of AuNps.

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Citrate ion capping on AuNps was also characterized using FTIR spectroscopy

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(Supplementary materials Fig. S3). In pure trisodium citrate, the asymmetric and symmetric

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stretching bands for the carboxylate groups appear at 1591 and 1399 cm-1 [39, 40]. In our

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synthesized AuNps, the asymmetric stretching band of the carboxylate shifted towards higher wave number (1620 cm-1). Whereas, the symmetric stretching band was noticed at slightly

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lower wave number (1385 cm-1) as compared to pure citrate. Both the observations match

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with an earlier report [39] and suggest an efficient capping of citrate on AuNps. X-ray diffraction (XRD) was also used to investigate the crystalline nature of AuNps. The

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XRD pattern is shown in Supplementary materials Fig. S4. Au nanocrystals had demonstrated

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four distinctive peaks at 2θ values of 38.2°, 44.4°, 64.6° and 77.5°. These peaks are expected to be originated from Bragg reflections by (111), (200), (220) and (311) in a face centered cubic (fcc) lattice. The pattern shown in X-ray diffractograph resembles very closely with earlier reports [41, 42].

3.2 Thioflavin T (ThT) fluorescence Enhancement in the fluorescence of ThT due to its binding with fibrils is a common marker for the detection of amyloids as reported earlier [43, 44]. Earlier, we had observed that the ThT fluorescence intensity does not change on addition of native HGD to ThT [12]. But the

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Journal Pre-proof emission intensity at 485 nm was found to be enhanced considerably when the incubated (at 37°C, pH 3, 20 h) solution of HGD was added to ThT. This confirms the fibrillation of HGD under the incubation conditions. This is also consistent with the earlier reports on the fibrillation of γC- and γD-crystallins under low pH incubation [5-8, 12, 45].

Furthermore, fibrillation inhibition efficiency of AuNps was studied at different concentrations of the nanoparticles. ThT emission intensity (at 485 nm) was highest for the

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fibrils of HGD made in absence of AuNps. With increase in the concentration of AuNps co-

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incubated with HGD, the ThT emission intensity was decreased (Fig. 2A). But, AuNps alone

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did not affect the fluorescence spectra of ThT (Supplementary materials Fig. S5). This clearly

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indicates the decrease in the fibrillar content of HGD in the presence of AuNps. The percentage inhibition of fibrillation was calculated for different concentrations of AuNps

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(Supplementary materials Table S1), which was found to be increased with increase in the

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50 40 30 20 10 0

0

1

2

3

4

5

% Inhibition of fibrillation

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A

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Fluorescence intensity at 485 nm

concentration of AuNps and finally reached to saturation level at ~6 nM of AuNps (Fig. 2B). 100 B 80 60 40 20 0

6

2

Concentration of AuNps (nM)

4

6

Concentration of AuNps (nM)

Fig. 2: (A) Histogram for ThT (50 μM) fluorescence intensity (at 485 nm) on addition of HGD fibrillar solutions made in the presence of different concentrations of AuNps (0-6.1 nM); λex: 442 nm. (B) Percentage inhibition of HGD fibrillation by different concentrations of AuNps. The line is drawn merely as a visual guide.

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Journal Pre-proof We had also studied the size effect of the nanoparticles on their inhibitory potential against fibrillation of HGD. AuNps with different sizes had been prepared by varying the concentration of the capping agent sodium citatre. The sizes were measured using dynamic light scattering (Supplementary materials Fig. S6 and Table S2). It has been noticed that AuNps having the sizes from 10-20 nm effectively inhibited amyloid fibrillation of HGD. On the other hand, the nanoparticles having size ~ 25 nm or higher were found to be much less

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effective inhibitor (Supplementary materials Fig. S7).

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3.3 Tryptophan fluorescence study

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Generally, amyloid fibrillation proceeds along with structural changes in the protein molecule

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due to partial unfolding of the native state of the protein. In case of HGD, this also results substantial changes in the microenvironment surrounding the tryptophan (Trp) residues as

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reported earlier [12]. The intrinsic tryptophan fluorescence of the protein depends on the

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surrounding polarity of the Trp residues. Trp emission intensity of native HGD was enhanced upon fibrillation and the λmax of the emission spectra had experienced a red shift from 323 to

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329 nm (Fig. 3). This suggests that the polarity around the Trp residues of HGD had

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increased due to fibrillation. The observation typically indicates that the fibrillation precedes through self-assembly formation by the partially unfolded protein molecules as partial unfolding increases the exposure of Trp residues towards solvent. Furthermore, Trp intrinsic fluorescence of HGD fibrils was decreased when the fibrils were made in the presence of 4 nM AuNps. In that case, a blue shift (from 329 to 325 nm) in the λmax was also noticed. This is more close to the fluorescence spectra of native HGD and indicates inhibition of amyloid fibrillation of HGD by the nanoparticles.

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Fluorescence intensity

Journal Pre-proof NHGD HGDf HGDf+AuNps

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200

0

350

400

450

Wavelength (nm)

Fig. 3: Trp fluorescence spectra (λex: 295 nm) of native HGD (NHGD) and HGD fibrils made

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in absence (HGDf) and presence of 4 nM AuNps (HGDf+AuNps).

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3.4 ANS binding study

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ANS (a fluorescent dye) is used widely to probe the hydrophobic surfaces in protein molecules. On binding of ANS with the hydrophobic patches of proteins, a blue shift in its

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emission maximum is generally observed along with an increase in its emission intensity. On

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addition of native HGD to ANS, the fluorescence spectrum of ANS was not changed. But, a dramatic increase in the emission intensity of ANS was noticed on addition of HGD fibrils

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(made as above) to a solution of ANS (20 μM). A remarkable blue shift in the emission λmax

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from 520 to 478 nm was also recorded (Fig. 4). This indicates that the hydrophobic patches of HGD (usually remain buried in native protein) were exposed due to fibrillation through partial unfolding of the protein. When HGD fibrils were made through co-incubation with 4 nM of AuNps, a notable diminution in ANS emission intensity was noticed on addition of such fibrillar solution to ANS. Furthermore, the observed blue shift in emission λmax for the HGD fibrils in the presence of AuNps was also less as compared to the HGD fibrils prepared in absence of AuNps. In the blank set, it was also noticed that there is no change in the fluorescence spectra of ANS on addition of 4 nM of the AuNps (incubated alone at 37˚C for 20 hours at pH 3.0) to ANS. These observations mechanistically suggest that AuNps

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Journal Pre-proof probably bind with the hydrophobic patches on the surface of native HGD, which are most likely to be exposed during partial unfolding of the protein leading to fibrillation. That is why co-incubation of HGD with AuNps, the surface hydrophobicity is much less as compared to

NHGD HGDf HGDf+AuNps 20M ANS

500 400

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300 200 100 0 400

450

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Fluorescence intensity

the HGD fibrils made in absence of AuNps.

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550

600

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Wavelength (nm) Fig. 4: Fluorescence spectra (λex: 380 nm) of ANS on addition of native HGD (NHGD),

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HGD fibril formed in absence (HGDf) and the presence of 4 nM of AuNps (HGDf+AuNps).

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3.5 Circular dichroism (CD)

As fibrillation of HGD involves major structural changes, therefore, the inhibition of HGD

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fibrillation by AuNps was also studied by using far-UV CD spectroscopy. AuNps did not

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exhibit any CD signal in this wavelength region. The CD value (in mdeg) at ~218 nm of HGD was found be increased due to the fibrillation of the protein (Fig. 5). This suggests a decrease in the β-sheet content of the protein, which become unordered during fibrillation through partial unfolding. When HGD fibrils were made in the presence of AuNps, the CD value at ~218 nm was again decreased. But the CD spectrum was not recovered completely with respect to native HGD. This infers that the secondary structural changes in HGD due to fibrillation are not fully reversible. Though AuNps were able to preserve the secondary structures of HGD to some extent, but the protein cannot be recovered back fully to its native structure by the nanoparticles.

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Journal Pre-proof NHGD HGDf HGDf+AuNps

CD (mdeg)

20 10 0 -10 -20 200

220

240

Wavelength (nm)

Fig. 5: CD spectra of native HGD (NHGD) and HGD fibril (HGDf) formed in absence and

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the presence of 4 nM of AuNps (HGDf+AuNps).

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3.6. Fluorescence microscopy and HRTEM

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Fluorescence microscopy was used to visualize ThT-stained HGD fibrils made in absence and the presence of AuNps. In Fig. 6A, highly fluorescent fibrils of HGD were found

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prominently, which were made in absence of AuNps. But in the presence of 4 nM of AuNps,

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fluorescent fibrils were found almost rarely (Fig. 6B), which demonstrates the antiamyloid activity of AuNps against fibrillation of HGD. The HRTEM image (Fig. 6C) reveals the

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formation of HGD fibrils in absence of AuNps. In the presence of 4 nM of AuNps, extent of

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HGD fibrillation was decreased significantly (Fig. 6D). Fluorescence and HRTEM microscopic images were found to agree with the spectroscopic results, which suggest that AuNps are able to inhibit amyloid fibrillation of HGD in an effective manner.

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Journal Pre-proof HGDf

B

HGDf+AuNps

C

HGDf D

HGDf+AuNps

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Figure 6: (A & B) Fluorescence microscopic and (C & D) TEM images of HGD fibrils made

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nm (C & D) respectively.

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in absence and the presence of 4 nM of AuNps. Scale bars represent 50 μm (A & B) and 200

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Amyloid inhibition of γ-crytallins is not reported much in the literature and only two earlier reports had been found [12, 13]. The inhibitory potential of AuNps with those reported inhibitors was compared. To achieve a similar inhibition, both the earlier works had reported that micromolar concentrations of inhibitors were required. In the present work, we had achieved the same extent of amyloid inhibition by using only nanomolar concentration of AuNps. Furthermore, to understand the process of inhibition of HGD fibrillation by AuNps, interactions between HGD and AuNps were studied at molecular level. Multispectroscopic techniques have been used to elucidate protein-nanoparticles interactions.

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Journal Pre-proof 3.7 Quenching of Trp fluorescence of HGD by AuNps Quenching of Trp intrinsic fluorescence of HGD at different concentrations (0-0.55 nM) of AuNps was studied at 294K (Fig. 7). Similar experiments were also performed at other temperatures 300, 305 and 310K (Supplementary materials Fig. S8). HGD being a multitryptophan protein had exhibited an emission maximum at ~324 nm. Addition of AuNps to HGD resulted considerable quenching of the fluorescence intensity of the protein. This indicates the close proximity of AuNps to the fluorophores of HGD. The emission maximum

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of HGD was not found to be altered on addition of AuNps. This suggests that the polarity of

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the Trp micoenvironment of native HGD was not changed due to the interactions with

0 nM

400

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-p

600

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Concentration of AuNps

0.55 nM

200

320

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0

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Fluorescence intensity

AuNps.

360

400

Wavelength (nm)

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Fig. 7: Fluorescence spectra of HGD (4 μM) in absence (apex line) and in presence of various concentrations of AuNps (0-0.55 nM) at 294K. λex: 295 nm and the arrow indicates the lowering of fluorescence intensity with increasing concentration of AuNps.

Using the quenching data at different concentrations of quencher (Q), Stern-Volmer plots (equation 1) [35] were drawn at four different temperatures. The plots deviate from linearity with an upward curvature and concave towards Y-axis (Fig. 8A). This suggests the probability of either combined (static and dynamic) quenching or the presence of different types of fluorophores with uneven accessibility [35]. Considering the uneven accessibilities

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Journal Pre-proof of the tryptophans, the modified form of the Stern-Volmer equation (equation 2) [35] was further used to analyze the quenching data (Fig. 8B). By using that, the quenching constant (KSV) and the fractional accessibility (fa) of the fluorophores were calculated at those temperatures. The value of fa greater than unity suggests the simultaneous presence of two accessible fractions corresponding to the two fluorophores of the protein at two different binding sites. The increasing values of fa with increase in temperature indicate that the

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fluorophores of HGD become more accessible to AuNps at higher temperatures.

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(1)

F0 1 1   F0  F f a K SV [Q] f a

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(2)

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Where F0 and F are the fluorescence intensities of HGD before and after addition of AuNps.

A

1.5

0.2

0.4

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1.0 0.0

F0/(F0-F)

lP na

2.0

B

294 K 300 K 305 K 310 K

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F0/F

2.5

25 20

294 K 300 K 305 K 310 K

15 10 5 0 0

0.6

[AuNps] nM

10

20

30

1/[AuNps] nM

40 -1

Fig. 8: (A) Stern-Volmer plots for quenching of Trp fluorescence of HGD (4 μM) by AuNps (0-0.55 nM) at 294, 300, 305 and 310K. (B) Modified Stern-Volmer plots for the same at those temperatures.

The decrease in quenching constant with rise in temperature is indicative of static quenching process in which HGD-AuNps complex is likely to be formed in the ground state. Furthermore, to elucidate the type of quenching process, bimolecular quenching constant (kq)

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Journal Pre-proof , where τ0 is the

values at those temperatures were calculated using the relation

fluorescence life time (~3 ns) of the significant fluorophores of HGD [46]. The values of KSV, fa and kq are given in Table 1. The maximum possible value of the collision quenching constant for biomacromolecules is 1 × 1010 M-1s-1 in aqueous medium [35]. But the kq values obtained in our case were found to be ~108 times higher than its largest possible value. This confirms that the observed fluorescence quenching is definitely of static nature and ground-

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state complex formation had occurred [35].

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Even if we assume that both the types of quenching were going on, then the Stern-Volmer

)

(

)

(3)

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=(

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equation can be written as (3) [35]

can also be written as

[

]

)

(4a) (4b)

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Therefore,

(

, where

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=

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Where, KD and KS are the dynamic and static quenching constants respectively. Equation (3)

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Where, Kapp is the apparent quenching constant. Using equation (4b), the values of Kapp at different concentrations of quencher were calculated using the fluorescence intensities before and after quenching at those quencher concentrations. If Kapp values are plotted against [Q] according to equation (4a), the values of static and dynamic quenching constants will be obtained from the slope and intercept of that plot (Fig. 9A). But, this resulted imaginary values of both the quenching constants, which proves that our assumption for equation (3) was wrong and the existence of combined quenching mechanism was thereby nullified.

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Journal Pre-proof The binding constant (K) and the number of binding sites (n) were also calculated (Table 1) using double logarithmic plots (equation 5) [36]. These plots at four different temperatures are shown in Fig. 9B. (

)

(5)

High value of binding constant (in the order of 109 M-1) for HGD-AuNps complex suggests strong binding between HGD and AuNps. The binding constant values were found to be

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increased slightly with increase in temperature. This suggests the possibility of hydrophobic interactions between HGD and AuNps.

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A

B

2.6

re

log (F0-F)/F

2.4

Kapp

294 K 300 K 305 K 310 K

-p

0.0

2.2

lP

2.0 1.8 0.2

0.4

[AuNps] nM

0.6

na

0.0

-0.5

-1.0

-1.5

-1.5

-1.0

-0.5

log [AuNps]

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Fig. 9: (A) The plot between Kapp and the concentration of AuNps. (B) Double logarithmic

0.55 nM.

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plots at four different temperatures. For both the plots, [HGD] = 4 μM and [AuNps] = 0 to

Table 1: Quenching and binding parameters for HGD-AuNps Temp. (K) 294 300 305 310

KSV (109 M-1)

fa

kq (1018 M-1s-1) K (x 109 M-1)

n

1.22±0.04 1.13±0.03 1.05±0.03 0.97±0.02

1.39±0.05 1.55±0.07 1.70±0.06 1.89±0.10

0.41±0.02 0.38±0.01 0.35±0.01 0.32±0.01

1.10±0.05 1.11±0.03 1.11±0.04 1.11±0.05

19

1.44±0.03 1.48±0.03 1.51±0.02 1.55±0.02

Journal Pre-proof From the above discussion, it can be concluded in a nutshell that the quenching mechanism for Trp fluorescence of HGD was static in nature. Combination of static and dynamic quenching mechanisms was also discarded. Ground state complexation between the protein and AuNps had basically caused quenching of HGD fluorescence. Steady state fluorescence also ruled out the possibility of major conformational changes of the fluorophores due to interactions with AuNps.

of

Various intermolecular forces like hydrogen bonding, hydrophobic interactions, van der

ro

Waals forces and electrostatic interactions are usually involved in the binding process of a

-p

ligand with a protein molecule. The sign and magnitude of the changes in enthalpy (∆H),

re

entropy (∆S) and free energy (∆G) associated with the interaction process indicate the nature of the binding forces involved between the protein and ligand [47]. These thermodynamic

lP

parameters (Table 2) for HGD-AuNps complexation were calculated using van’t Hoff plot

ur

Jo

lnK

21.15

na

(Fig. 10).

21.12

21.09 0.0032

0.0033 -1 1/T (K )

0.0034

Fig. 10: van't Hoff plot for the binding of HGD with AuNps. [HGD] = 4 μM.

20

Journal Pre-proof Table 2: Thermodynamic parameters for binding of AuNps with HGD Temperature (K)

∆G (kJ mol-1)

294

-51.54±0.42

300

-52.66±0.48

305

-53.60±0.51

310

-54.53±0.55

∆H (kJ mol-1)

∆S (Jmol-1K-1)

3.44±0.22

187±6.2

Spontaneous complexation between HGD and AuNps was confirmed by negative ∆G value.

of

For HGD-AuNps complexation, the major contribution to the ∆G values came from ∆S.

ro

Thus, the binding of AuNps with HGD is mainly driven by the entropic factor. The positive

-p

changes both in enthalpy and entropy along with dependence of the process solely on the

lP

association of AuNps with HGD [47].

re

entropic factor suggest that hydrophobic interactions play the most significant role during the

3.8 Circular dichroism

na

Far-UV CD of native HGD (NHGD) in absence and presence of AuNps were recorded to

ur

monitor conformational changes in HGD due to its interactions with AuNps (Fig. 11). Only a

Jo

slight variation was observed, which indicates non-significant changes in the global conformation of the protein due to interactions with AuNps.

21

Journal Pre-proof

CD (mdeg)

20

NHGD NHGD+AuNps

10 0 -10 -20 200

220

240

Wavelength (nm) Fig. 11: CD spectra of native HGD in absence (NHGD) and the presence of 4 nM of AuNps

ro

of

(NHGD + AuNps). [HGD] = 0.35 mg/ml

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3.9 Synchronous fluorescence

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Synchronous fluorescence studies give information about the microenvironment in the neighborhood of the fluorophores present in a protein. Characteristic information about Trp

lP

residues are obtained when the scanning interval (Δλ) between the excitation and emission

na

monochromator is fixed at 60 nm. The effect of AuNps on the synchronous fluorescence spectra of HGD is shown in Fig. 12. The synchronous fluorescence intensity decreases

ur

gradually on continuous addition of AuNps, but no substantial changes arise in emission

Jo

maxima of HGD. This suggests that no significant change occurs in the polarity around the Trp residues due to HGD-AuNps complex formation.

22

Fluorescence intensity

Journal Pre-proof 500

0 nM

concentration of AuNps

400 300

0.5 nM

200 100 300

350

400

Wavelength (nm)

Fig. 12: Synchronous fluorescence spectra of HGD in absence (apex line) and in presence of

ro

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AuNps for Δλ = 60 nm. [HGD] = 4 μM and [AuNps] = 0-0.5 nM.

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4 Conclusions

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Gold nanoparticles (AuNps) were synthesized to achieve inhibition in HGD fibrillation. AuNps were characterized using UV-Vis spectroscopy, dynamic light scattering, FTIR and

lP

XRD. The average diameter of AuNps was found to be ~14 nm. ThT fluorescence assay

na

indicated substantial fibrillation of HGD at pH 3.0 for 20 hours at 37˚C in absence of AuNps and that was suppressed by AuNps at nanomolar concentrations. Exposure of hydrophobic

ur

surfaces of HGD due to its fibrillation was also reduced in the presence of AuNps as

Jo

suggested by ANS binding assay. The decrease in the hydrophobic exposure of HGD during fibrillation in presence of AuNps explains the inhibition mechanism. Microscopic images revealed that the fibrillar structures of HGD were formed and AuNps can decrease the fibrillar content. Studies on the interactions between native HGD and AuNps suggest strong binding between HGD and AuNps. Static quenching of HGD Trp fluorescence was observed. No major conformational changes were observed in native HGD due to its binding with AuNps. Thermodynamic parameters revealed the major involvement of hydrophobic interactions in the association process between AuNps and HGD.

23

Journal Pre-proof Acknowledgements: KSG is grateful to the Director, NIT Hamirpur for providing necessary infrastructural facilities to accomplish this work. KSG is also grateful to Prof. D. Balasubramanian, L.V. Prasad Eye Hospital, Hyderabad for his kind gift of HGD clone. Authors are also thankful to Dr. Rajanish Giri, IIT Mandi and Dr. Tushar Kanti Maiti, Regional Center for Biotechnology, Faridabad for providing support in fluorescence microscopy and CD respectively. The supports from Department of Material Science and Engineering, NIT Hamirpur for DLS,

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AMRC, IIT Mandi and NIPER, Mohali for TEM imaging are gratefully acknowledged.

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Journal Pre-proof

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Authors contribution The work was designed by KSG and VS. Experimental data were acquired by VS, SS and SR. Analysis and/or interpretation of the experimental results, drafting and further revision of the manuscript were done by all the authors. Graphical abstract

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Highlights Inhibition of HGD fibrillation by AuNps



Static quenching of Trp fluorescence of HGD by AuNps



Binding constant for HGD-AuNps in the range of 109 M-1



No notable conformational change in HGD due to its interactions with AuNps



Hydrophobic interactions derive the binding between AuNps and HGD

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lP

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

31