Selective inhibition of aggregation/fibrillation of bovine serum albumin by osmolytes: Mechanistic and energetics insights

RESEARCH ARTICLE Selective inhibition of aggregation/fibrillation of bovine serum albumin by osmolytes: Mechanistic and energetics insights Moumita Dasgupta, Nand Kishore* Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai, India * Abstract a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Dasgupta M, Kishore N (2017) Selective inhibition of aggregation/fibrillation of bovine serum albumin by osmolytes: Mechanistic and energetics insights. PLoS ONE 12(2): e0172208. doi:10.1371/journal.pone.0172208 Editor: Eugene A. Permyakov, Russian Academy of Medical Sciences, RUSSIAN FEDERATION Received: December 29, 2016 Accepted: February 1, 2017 Published: February 16, 2017 Copyright: © 2017 Dasgupta, Kishore. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Support was provided by the Indian Institute of Technology, Bombay. Competing interests: The authors have declared that no competing interests exist. Bovine serum albumin (BSA) is an important transport protein of the blood and its aggregation/fibrillation would adversely affect its transport ability leading to metabolic disorder. Therefore, understanding the mechanism of fibrillation/aggregation of BSA and design of suitable inhibitor molecules for stabilizing its native conformation, are of utmost importance. The qualitative and quantitative aspects of the effect of osmolytes (proline, hydroxyproline, glycine betaine, sarcosine and sorbitol) on heat induced aggregation/fibrillation of BSA at physiological pH (pH 7.4) have been studied employing a combination of fluorescence spectroscopy, Rayleigh scattering, isothermal titration calorimetry (ITC), dynamic light scattering (DLS) and transmission electron microscopy (TEM). Formation of fibrils by BSA under the given conditions was confirmed from increase in fluorescence emission intensities of Thioflavin T over a time period of 600 minutes and TEM images. Absence of change in fluorescence emission intensities of 8-Anilinonaphthalene-1-sulfonic acid (ANS) in presence of native and aggregated BSA signify the absence of any amorphous aggregates. ITC results have provided important insights on the energetics of interaction of these osmolytes with different stages of the fibrillar aggregates of BSA, thereby suggesting the possible modes/ mechanism of inhibition of BSA fibrillation by these osmolytes. The heats of interaction of the osmolytes with different stages of fibrillation of BSA do not follow a trend, suggesting that the interactions of stages of BSA aggregates are osmolyte specific. Among the osmolytes used here, we found glycine betaine to be supporting and promoting the aggregation process while hydroxyproline to be maximally efficient in suppressing the fibrillation process of BSA, followed by sorbitol, sarcosine and proline in the following order of their decreasing potency: Hydroxyproline> Sorbitol> Sarcosine> Proline> Glycine betaine. Introduction The aggregation of proteins can take place under various environmental conditions, giving rise to higher order supramolecular structures including fibrillar forms [1,2]. The phenomenon could be either, temperature, pH or concentration driven, or combination of these said factors. The major pathways of protein aggregation are–(i) through the partially folded states PLOS ONE | DOI:10.1371/journal.pone.0172208 February 16, 2017 1 / 26 Qualitative and quantitative insights into prevention of BSA fibrillation by osmolytes or intermediates, (ii) self association of protein molecules, (iii) chemical linkages or via chemical degradation [1]. The rationale behind the aggregation pathways are the net surface charge that could be repelling as well as the hydrophobic effect which gives rise to attractive forces between the monomers [3]. In case, where the aggregation occurs due to high temperature, the hydrophobic interactions play significant role rather than electrostatic interactions [4]. Again at a particular temperature and pH, the concentration determines the size of the aggregates and variation of concentration under such conditions could bring about formation of oligomers to gel from those aggregates. The aggregation pathways are complex enough to give rise to the formation of aggregates of diverse morphologies based on solution conditions and above mentioned physical factors [3]. Amyloid fibrils are categorized into one of those morphologies and are associated with neurodegenerative diseases, while the prefibrillar aggregates are the causative agents of cytotoxic effects in vivo [2,3]. Structurally, the protein aggregates are found to be β-sheet rich, where amyloids are typical cross β structures that are aligned perpendicularly to the axis of the fibril [3]. However, in recent time, active research is going on, in order to implement these fibrillar structures, generated in vitro, in the field of biomedicine and bionanotechnology [5]. These fibrils find their application from development of peptide nanotubes useful in bio-electrochemical sensor applications to creation of scaffolds applicable in tissue engineering and drug delivery [4]. The fibrils could also be used for bacterial biofilm development [2]. However, inhibition of protein aggregation in vivo, is extremely important in order to prevent and control the occurrence of neurodegenerative diseases. Herein lies the importance of studying the effect of external small molecules, that could potentially arrest the aggregation propensity of proteins and stabilize the native form. Our study can guide the development of therapeutic strategies against the diseases caused due to protein aggregation/fibrillation. The naturally occurring small molecules, called osmolytes, synthesized in various organisms in response to stress [6], are known to play crucial role in protecting cell against high osmotic condition by maintaining cell volume and fluid balance as well as increasing the thermodynamic stability of protein molecules thereby stabilizing the functional folded form. Hence the osmolytes are also known as “chemical chaperones” [6,7]. These organic compounds could be amino acids (e.g. proline, glycine and arginine), various polyols (e.g. sorbitol and sucrose) and methyl amines (e.g. betaine and trimethylamine-N-oxide) [6]. There are several reports on osmolyte mediated prevention of protein misfolding and aggregation [8–10]. The enhancement in thermal stability of proteins, brought about by the osmolytes, is known to be via the phenomenon of preferential hydration [8,9,11–13]. In the current study, an approach has been taken to explore and understand the aggregation behavior of bovine serum albumin (BSA) at physio (...truncated)