Inhibition of HSA Fibrillation by Two Dimensional Nanoparticles

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Inhibition of HSA Fibrillation by Two Dimensional Nanoparticles Sudipta Bag, Rishav Mitra, Sunando DasGupta, and Swagata Dasgupta J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 15 May 2017 Downloaded from http://pubs.acs.org on May 16, 2017

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The Journal of Physical Chemistry

Inhibition of HSA Fibrillation by Two Dimensional Nanoparticles

Sudipta Baga, Rishav Mitraa, Sunando DasGuptab, Swagata Dasguptaa* a

Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur 721302,

India b

Department of Chemical Engineering, Indian Institute of Technology Kharagpur, Kharagpur

721302, India

*Corresponding Author: Professor Swagata Dasgupta Department of Chemistry Indian Institute of Technology Kharagpur, Kharagpur 721302, India Tel.: +91-3222-283306 Email: [email protected]

1 ACS Paragon Plus Environment


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Abstract The formation and deposition of amyloid fibrils have been linked to the pathogenesis of numerous debilitating neurodegenerative disorders. Serum albumins serve as good model proteins for understanding the molecular mechanisms of protein aggregation and fibril formation. Graphene based nanotherapeutics appear to be promising candidates for designing inhibitors of protein fibrillation. The inhibitory effect of graphene oxide (GO) nanoparticles on the fibrillation of human serum albumin (HSA) in an in vitro mixed solvent system has been investigated. The methods used include ThT fluorescence, ANS binding, Trp fluorescence, circular dichroism, fluorescence microscopy, FESEM and HRTEM. It was observed that GO inhibits HSA fibrillation and forms agglomerates with β-sheet rich prefibrillar species. Binding of GO prevents the formation of mature fibrils with characteristic cross-β sheet but does not promote refolding to the native state.


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1. Introduction Protein folding pathways are interspersed with stochastic misfolding errors that can often lead to the formation of aggregation-prone partially unfolded states. These in turn may selfassemble to form oligomeric structures which could finally lead to the formation of amyloid fibrils.1 Amyloid fibrils have been linked to numerous protein deposition diseases like Alzheimer’s, Parkinson’s, Huntington's diseases, prionopathies, Type 2 diabetes, etc.2,3 The amyloid fibrils are a special type of insoluble ordered protein deposits that possess a generic core crossed β-sheet structure.4 The choice between the three aggregation pathways starting with an ensemble of ‘‘sticky’’ aggregation-prone intermediates: fibrillation, amorphous aggregate formation or oligomerization, is determined by the amino acid sequence and the physicochemical environment of the protein under in vitro conditions.5 Many folded proteins can undergo amyloid fibril formation under specific in vitro conditions that promote destabilization of the native state, such as elevated temperature,6,7 pH,8–10 high pressure,11 the presence of salts,12,13 metal ions14,15 cosolvents,16 surfactants,17 and nanoparticles.18 Human serum albumin (HSA) is the most abundant serum protein (∼60%) and is widely used as a model all α-helical protein to gain mechanistic insights into protein folding, misfolding and aggregation. HSA performs several important functions in the blood which include the transport and distribution of a wide range of physiologically relevant molecules like hormones, nutrients, fatty acids, drugs, maintenance of blood colloidal osmotic pressure, etc.19 It is an α-helical globular protein consisting of 585 amino acid residues having three domains, I, II, and III each of which consist of two subdomains, a and b with common structural motifs. The sole tryptophan residue (Trp 214) of HSA lies in subdomain IIa.20 The ordered aggregation of globular proteins like HSA requires the partial unfolding of the native structure which exposes aggregation prone regions in the protein.21–25 Mature amyloid fibrils



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of HSA show morphological variability due to conformational switching to alternate aggregation pathways under different solution conditions.23 Bovine serum albumin (BSA) also undergoes fibrillation under various pH conditions and is reported to follow a complex, “lag-phase-independent”, nucleated conformational conversion mechanism.26 In this mechanism, the native state expands at low pH to form a molten globule-like state which aggregates to form an apparent conformationally converted “propagation competent nucleus” that promotes fibril assembly.25 Formation of HSA fibrils can be achieved using various methods like lowering the pH of the medium, addition of denaturants, and use of organic solvents etc. Alcohols destabilize the hydrophobic interactions, thereby denaturing the native fold of proteins. The effect of the mixed alcohol-water system in the formation of amyloid fibrils of HSA has also been previously investigated including studies from this laboratory.27,28 Nanomaterials are becoming increasingly important in several fields of biomedical applications that include drug delivery, encapsulation and drug design etc. The characteristic nanoparticle composition, functional groups, shape and size are found to play significant roles in interactions with biomolecules.29–32 Literature shows that nanoparticles have both inhibitory and stimulatory activity in the process of amyloid fibrillation.29–34 We have recently shown that amine functionalized magnetic nanoparticles show inhibition of HSA fibrillation in vitro.35 Graphene is a planar, single atom thick two dimensional nanomaterial which shows excellent electrical, thermal and mechanical properties.36 Chemical modification of graphene is important for increasing the water solubility of the material for biomedical applications. Graphene oxide (GO), chemically modified graphene is decorated by reactive oxygen functional groups like hydroxyl, epoxy groups etc. Carboxylic groups are also found to be present at the periphery of the planar surface. The water solubility of GO is attributed to the


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presence of these groups, thereby enhancing its biomedical applications.37 Previously, GO has shown potential applications in the inhibition of amyloid β-peptide fibrillation.38 Interactions of GO with native serum albumins as well as amino acids have been reported earlier.39,40 However, to the best of our knowledge, effects of GO on the fibrillation of HSA have not been investigated. In the present article, we have studied the effects of GO on the fibrillation of HSA using biophysical techniques. Graphene oxide was synthesized following a modified Hummer’s method41,42 and characterized by different spectroscopic and microscopic approaches. The effects of GO on HSA fibrillation were studied using fluorescence spectroscopy, circular dichroism spectroscopy. Microscopic analyses were performed to examine morphological changes of HSA fibrils upon incubation with GO.

2. Materials and Methods 2.1. Materials Thioflavin

T

(ThT), graphite powder of size

Inhibition of HSA Fibrillation by Two Dimensional Nanoparticles

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