Enhancement in chaperone activity of human αA-crystallin by nanochaperone gold nanoparticles: Multispectroscopic studies on their molecular interactions

https://doi.org/10.1016/j.saa.2022.121344Get rights and content

Highlights

Abstract

The chaperone activity of human αA-crystallin (HAA) against aggregation of human γD-crystallin (HGD) was enhanced by gold nanoparticles (AuNPs). Chaperone activity of HAA was almost doubled in the presence of 5.5 nM gold nanoparticles (AuNPs). To decipher this effect at molecular level, interactions between HAA and AuNPs were studied using fluorescence and circular dichroism spectroscopic techniques. The nanoparticles were synthesized and characterized by using TEM and dynamic light scattering (DLS). TEM and DLS studies revealed that bioconjugation of AuNPs with HAA did not cause any significant change in the size of the nanoparticles. AuNPs had caused static quenching of tryptophan (Trp) fluorescence, which was confirmed through determination of excited state lifetime of Trp residue of HAA in absence and the presence of AuNPs. The association and quenching constant for HAA-AuNPs conjugation were ∼ 109 M−1. Hydrogen bonding and van der Waals interactions were found to be involved in HAA-AuNPs complex. Polarity of Trp microenvironment in HAA was not perturbed by AuNPs as supported by synchronous and three-dimensional fluorescence spectroscopy. Far-UV CD spectra suggested that the secondary structure of HAA was not significantly affected by AuNPs.

Introduction

The bulk of the eye lens is made of firmly packed fibres cells, which are devoid of some major organelle. For proper vision, light entering in the lens through cornea is transmitted and focused on the retina. The lens fibre cells involved in the above process are mostly comprised of crystallin proteins. These proteins are present at a high concentration and account for the transparency of lens. Crystallins are structural proteins and categorized in to three types (α, β and γ). Alpha-crystallin possesses chaperone-like behaviour [1], [2], [3]. Alpha-crystallin contributes ∼ 28% to the total protein content of the lens and maintains the required refractive index. Two subunits of α-crystallin (αA and αB) exist in 3:1 proportion [4]. Under stressful conditions, partial unfolding of β and γ-crystallins cause their aggregation. As a molecular chaperone, α-crystallin forms stable complexes with partially unfolded β and γ-crytallins to suppress their aggregation [5]. However, with aging, the chaperone activity of α-crystallin deteriorates and accumulation of protein aggregates begins in the lens. In normal condition, the metal ions like Cu2+ and Zn2+ are kept bound by α-crystallin [6], which suppresses metal-mediated oxidative protein aggregation in the lens [7]. But complexation between α-crystallin and its substrates (β and γ-crystallins) in aged lens deprive the metal binding ability of α-crystallin [8]. In that situation, β/γ-crystallins start to form protein aggregates, which scatter light and increase the opacity of the lens [9], [10]. This is commonly referred as cataract and recognized as the main cause of blindness worldwide. The aggregates thus formed remain in the lens for lifetime and can only be removed surgically.

An alternate non-surgical approach can be developed by increasing the chaperone activity of α-crystallin in aged lens which would delay the formation of cataract. Attempts to enhance the chaperone activity of α-crystallin had been reported earlier using small molecules, antiglycating agents etc. [11]. Earlier our group had also reported a synthetic Schiff base molecule having positive cooperative effect on the chaperone activity of human αA-crystallin (HAA) [12]. Further, molecular interactions between HAA and that Schiff base were studied to find out the mode of action [13]. Application of nanoparticles in the augmentation of the chaperone activity of αA-crystallin would be a remarkable feat in prolonging the development of cataract. In the present work, gold nanoparticles (AuNps) were used to enhance the chaperone activity of HAA. Selection of AuNPs in this purpose was based on earlier works, which had reported the potency of AuNPs to serve as nano-chaperone [14], [15], [16], [17], [18], [19]. Moreover, AuNPs was emerged as potent inhibitor against aggregation of different proteins and peptides [20]. Recently, our group had also reported the inhibition of amyloid fibrillation of human γD-crystallin (HGD) by AuNPs [21]. Based on these reports, application of AuNPs as nano-chaperone in the presence of HAA during aggregation of HGD through partial unfolding was attempted. Furthermore, interactions between HAA and AuNPs were studied.

Section snippets

Materials and methods

The reagents and culture media were purchased from Himedia and SRL. Absorption spectra were recorded on Hitachi UH5300 and fluorescence spectra on Shimadzu RF-5301PC and RF-6000. All the experiments were carried out in phosphate buffer (10 mM, pH 7.0). Over-expression and purification of HGD and HAA was accomplished following the procedure described earlier by Chauhan et al. [12].

Gold nanoparticles (AuNPs) were prepared from HAuCl4 (1 mM) through reduction by using tri-sodium citrate (20 mM)

Results and discussion

Gold nanoparticles (AuNPs) have earlier been reported as nanochaperone [14], [15], [16], [17], [18], [19]. The present work was thought with an idea to boost up the chaperone activity of HAA under the limiting conditions in an aged lens. Therefore, the effect of nanochaperone like AuNPs on the chaperone activity of HAA was checked. To assess the chaperone activity of HAA, aggregation of its substrate (HGD) was initiated during the refolding of the protein, which was previously unfolded by

Conclusions

Multispectroscopic studies on the interactions between HAA and AuNPs were presented in this work. Formation of ground-state complex between HAA and nanoparticles leads to static quenching of Trp fluorescence. Thermodynamic parameters suggest spontaneous bioconjugation. Hydrogen bonding and van der Waals interactions were involved in the complexation between HAA and AuNPs. The Trp microenvironment of HAA was not altered by AuNPs. The nanoparticles also did not perturb the secondary structure of

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

AS and KSG are thankful to the Director, NIT Hamirpur for providing research facilities. AS is obligated to NIT Hamirpur for Senior Research Fellowship. AS is thankful to Dr. Arvind Kumar and Dr. Tapas Palai (NIT Hamirpur), Prof. Pramit Kumar Chowdhury (IIT Delhi), Dr. Anjan Chakraborty and Avijit Maity (IIT Indore) for some instrumental supports.

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