Awanish Kumar

Does the stability of proteins in ionic liquids obey the Hofmeister series

Understanding the behavior of Hofmeister anions of ionic liquids (ILs) on protein stability helps to shed light on how the anions interact with proteins in aqueous solution and is a long standing object for chemistry and biochemistry. Ions effects play a major role in understanding the physicochemical and biological phenomenon that undertakes the protein folding/unfolding and refolding process. Despite the generality of these effects, our understanding of ions at the molecular-level is still limited. This review offers a tour through past successful investigations and presents a challenge in current research in the field to reassess the possibilities of ions and to apply new strategies. This review highlights on the stability behavior of the proteins and also comparisons of our past research work in the Hofmeister series of ILs. Furthermore, we specifically focus on the critical discussion on the recent findings with existing results and their implications, along with our understanding of the Hofmeister series of anions of ILs on biomolecular stability. A detailed examination of the difference between selective proteins can provide a better understanding of the molecular mechanism of protein folding/unfolding in the presence of the Hofmeister series of ions of ILs.

Prevention of insulin self-aggregation by a protic ionic liquid

The self-aggregation and thermal instability of insulin (In) was considerably controlled in the presence of ammonium-based protic ionic liquids (PILs). The thermal stability of In in PILs was observed using fluorescence and absorption spectroscopy of the Tyr environment of the biomolecule. Additionally, from circular dichroism (CD) measurements, we observed the shift in the wavelength towards lower values in the presence of PILs, which indicates the formation of monomers of In, further evidently supported by dynamic light scattering (DLS) measurements. Surprisingly, it is the monomeric form of the In that exists in the active form. For the first time, ammonium-based PILs have been shown to be novel solvents for In, which prevent it from associating into an inactive form and also stabilizes In against thermal influence.

The stability of insulin in the presence of short alkyl chain imidazolium-based ionic liquids

Fibril or aggregation formation in insulin (In) has been a subject of severe biomedical and biotechnological complications. The search for a novel solvent/co-solvent that can provide long term stabilization for In monomeric form has not been completed yet. In this quest, for the first time we have successfully explored the stability of the monomeric form of In in the presence of ammonium-based ionic liquids (ILs) [A. Kumar and P. Venkatesu, RSC Adv., 2013, 3, 362–367]. Further, in continuation, in this study, we have established the stability of In in the presence of imidazolium-based ILs with different anions. These anions represent the Hofmeister series of anions of ILs. In this regard, we have carried out UV-vis, fluorescence, circular dichroism spectral analysis and dynamic light scattering (DLS) measurements of In in various concentrations of these ILs. Our experimental findings reveal that Br− and Cl− ILs stabilized the native state while the rest of the ILs with anions such as SCN−, HSO4−, CH3COO− and I− were denaturants for the In. Further, the results show that IL–In interactions are difficult to classify on the basis of the Hofmeister series, as bromide containing ILs show more stabilizing properties on the In structure. The disulfide bonds were almost intact in the presence of Br− IL as compared to Cl− and the rest of the Hofmeister anions. On the other hand, a strongly hydrated kosmotropic anion like HSO4− interacts with the structure of In, leading it towards complete denaturation of the In structure. Additionally, all ILs failed to protect the native state of In with increasing temperature.

Destruction of hydrogen bonds of poly(N-isopropylacrylamide) aqueous solution by trimethylamine N-oxide

Trimethylamine N-oxide (TMAO) is a compatible or protective osmolyte that stabilizes the protein native structure through non-bonding mechanism between TMAO and hydration surface of protein. However, we have shown here first time the direct binding mechanism for naturally occurring osmolyte TMAO with hydration structure of poly(N-isopropylacrylamide) (PNIPAM), an isomer of polyleucine, and subsequent aggregation of PNIPAM. The influence of TMAO on lower critical solution temperature (LCST) of PNIPAM was investigated as a function of TMAO concentration at different temperatures by fluorescence spectroscopy, viscosity (η), multi angle dynamic light scattering, zeta potential, and Fourier transform infrared (FTIR) spectroscopy measurements. To address some of the basis for further analysis of FTIR spectra of PNIPAM, we have also measured FTIR spectra for the monomer of N-isopropylacrylamide (NIPAM) in deuterium oxide (D(2)O) as a function of TMAO concentration. Our experimental results purportedly elucidate that the LCST values decrease with increasing TMAO concentration, which is mainly contributing to the direct hydrogen bonding of TMAO with the water molecules that are bound to the amide (-CONH) functional groups of the PNIPAM. We believed that the present work may act as a ladder to reach the heights of understanding of molecular mechanism between TMAO and macromolecule.

Trehalose protects urea-induced unfolding of α-chymotrypsin.

Trehalose, a naturally occurring osmolyte, appears to be one of the most effective protectants for enzymes under various stress conditions while urea, a classical denaturant, destabilizes the activity, function, and alters the native structure of proteins. Herein, we have characterized the counteracting effects of trehalose on the deleterious effect of urea on α-chymotrypsin (CT) through the calorimetric data (transition temperature (T(m)), enthalpy change (ΔH), heat capacity change (ΔC(p)) and Gibbs free energy of unfolding (ΔG(u)) by using differential scanning calorimeter (DSC) and circular dichroism (CD) techniques, respectively, at a 1:2 ratio of trehalose and urea, as well as various urea concentration (up to 6 M) in the presence of 1 M trehalose. Our parallel experimental results explicitly elucidate that trehalose strongly offset the deleterious actions of urea on CT at 1:2 molar ratio of trehalose and urea, however, trehalose (1 M) some how failed to counteract the perturbation effects of urea (3-6 M) on CT.

A comparative study of the effects of the Hofmeister series anions of the ionic salts and ionic liquids on the stability of α-chymotrypsin

In this article, we have compared the anions of sodium salts (Is) and ionic liquids (ILs) with the stability and structure of α-chymotrypsin (CT), through fluorescence, thermal fluorescence analysis and circular dichroism (CD) spectroscopy. The experimental results revealed that the Hofmeister series of anions such as SCN−, SO42−, Cl−, Br−, CH3COO− and I− of Is destabilized the native structure of CT. On the contrary, the anions such as CH3COO−, Cl− and Br− of imidazolium-based IL with a fixed cation such as 1-butyl-3-methylimidazolium, [Bmim]+, stabilized the native structure of CT. The remaining anions of ILs such as SCN−, HSO4−, and I− acted as denaturing agents for the native structure of CT. Furthermore, molecular docking results show that the imidazolium-cation of the IL enters the sub-domains of CT and interacts with the ionic residues of CT, that is, Ser217 close to Trp215. This interaction is in well agreement with the fluorescence quenching observed for CT in the presence of [Bmim]+. On the other hand, the destabilizing anion such as SO42− was observed to be directly interacting with Ser195 in the active site of CT. We have observed that the Hofmeister series effects of anions of either Is or ILs are entirely based on the interaction of the anions with their counterions, that is, cations, with solvent molecules, as well as with the protein surface. Evidently, these interactions vary with the co-solvent system and the type of protein. Hence, the stability of a biomolecule in the presence of the anions may or may not obey the Hofmeister series.

The Overriding Roles of Concentration and Hydrophobic Effect on Structure and Stability of Heme Protein Induced by Imidazolium-Based Ionic Liquids

Spectroscopic and molecular docking investigations were carried out to characterize the effect of imidazolium-based ionic liquids (ILs) with varying chain length of the cation on the thermal stability as well as spectroscopic behavior of heme protein hemoglobin (Hb). The goal of this work is to investigate the role of concentration of ILs, the effect of alkyl chain length of the cation, and the related Hofmeister series on the structure of Hb. To achieve this goal, a series of ILs possessing same Cl(-) anion and a set of cation [Cnmim](+) with increasing chain length such as 1-ethyl-3-methylimidazolium chloride ([Emim][Cl]), 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]), 1-hexyl-3-methylimidazolium chloride ([Hmim][Cl]), and 1-decyl-3-methylimidazolium chloride ([Dmim][Cl]) were used in this study. It was observed that the stability of the protein was concentration dependent as well as the hydrophobic interactions between [Cnmim](+) of ILs, and the amino acid residues in the protein played a major role in protein unfolding. As a consequence, the destabilization tendency of the ILs toward the Hb increases with increasing chain length of the cation of ILs. Additionally, the cations of the ILs obeyed the Hofmeister series when arranged in the order of providing stability to Hb structure.

The biological stimuli for governing the phase transition temperature of the "smart" polymer PNIPAM in water.

A lack of sufficient knowledge regarding the behaviour of stimuli-responsive polymers to biological stimuli hinders the potential use of responsive polymers as biomaterials and medical devices. Hence, in this study, we demonstrate the impact of various globular proteins on the phase transition temperature of poly(N-isopropylacrylamide) (PNIPAM) in an aqueous solution through the use of fluorescence spectroscopy, dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy and field-emission scanning electron microscopy (FESEM). Furthermore, we describe the molecular interaction of PNIPAM with proteins by the MolDock method. Our experimental and docking studies revealed that such proteins as α-chymotrypsin (CT), insulin (In) and haemoglobin (Hb) decreased the lower critical solution temperature (LCST) of the polymer, whereas succinyl-concanavalin A (SCA) increased the LCST of PNIPAM. The LCST changed upon increasing the concentration of protein from 0.5mg/mL to 1mg/mL. The thermoresponsive behaviour of PNIPAM can be significantly altered by the functional groups present in the protein. The findings of the present study can be used in the engineering of bioresponsive smart PNIPAM-based devices.

Analysis of the driving force that rule the stability of lysozyme in alkylammonium-based ionic liquids.

Ionic liquids (ILs) have found various applications in the field of biotechnology that involves protein extraction from the aqueous phase. However, the stability of biomolecules in ILs is still unpredictable. Therefore, this work aims to understand the effect of ammonium-based ILs with a fixed (trifluoromethylsulfonyl)imide [NTf2](-) anion and variable ammonium cations such as butyltrimethylammonium (IL-1), ethyldimethylpropylammonium (IL-2), diethylmethyl(2-methoxyethyl)ammonium (IL-3) and methyl-trioctylammonium (IL-4) on the stability of lysozyme. The spectroscopic analysis (UV, fluorescence and circular dichroism (CD)) revealed the existence of native structure of lysozyme in the presence of ILs at 25°C. Evidently, the presence of α-helix structure in lysozyme was confirmed using CD spectroscopy. In contrary, the thermal stability of the protein gradually decreased with increase in the concentration of the ILs. This was due to the strong favorable interactions of the ILs with the amino acid residues of the protein. Further, Nile red fluorescence revealed existence of the hydrophobic interactions between ILs and the lysozyme. Hence, due to its immense hydrophobic character, IL-4 thereby, decreased the catalytic activity and stability of the lysozyme to a greater extent.

Exploring the structure and stability of amino acids and glycine peptides in biocompatible ionic liquids

Amino acids (AAs) are vital components for a variety of biological systems and can be linked through covalent bonds (or peptide bonds) to form a protein structure. Essentially, the interactions of these AAs with solvents as well as co-solvents/solutes determine the thermodynamic stability of the protein. In this context, this review represents an overview of the current status of the thermodynamic effect of ionic liquids (ILs) on AAs and glycine peptides (GPs). Moreover, ILs are considered as green solvents for many chemical and biological processes due to their tunable physical properties. Interestingly, these ILs can adjust themselves in any required experimental conditions such as protein extraction to enzyme catalysis. In this review, we attempt to assess the status of our current understanding on the biocompatible nature of the ions of ILs on AAs and protein model compounds and their functional groups with some precisely available experimental data in the literature. Examples of current applications of the ILs on protein model compounds are also covered in this review.