Indrani Jha

Unexpected effects of the alteration of structure and stability of myoglobin and hemoglobin in ammonium-based ionic liquids

The nature of solvent-biomolecule interactions is generally weak and non-specific. The addition of ionic liquids (ILs), which have emerged as a new class of solvents, strengthen the stability of some proteins whereas the same ILs weaken the stability of some other proteins. Although ILs are commonly used for the stabilization of biomolecules, the bimolecular interactions of their stabilization-destabilization is still an active subject of considerable interest and studies on this topic have been limited. To reveal the impact of ILs on the stability of proteins, a series of protic ILs possessing a tetra-alkyl ammonium cation [R4N](+) with a hydroxide [OH](-) anion were synthesized. In this study, we report the structural stability of heme proteins such as myoglobin (Mb) and hemoglobin (Hb) in a series of ammonium-based ILs such as tetramethyl ammonium hydroxide [(CH3)4N](+)[OH](-) (TMAH), tetraethyl ammonium hydroxide [(C2H5)4N](+)[OH](-) (TEAH), tetrapropyl ammonium hydroxide [(C3H7)4N](+)[OH](-) (TPAH) and tetrabutyl ammonium hydroxide [(C4H9)4N](+)[OH](-) (TBAH) by fluorescence and circular dichroism (CD) spectroscopic studies. Our experimental results reveal that less viscous ILs carrying smaller alkyl chain such as TMAH are strong destabilizers of the heme proteins as compared to the ILs carrying bulkier alkyl chains which are more viscous ILs, such as TBAH. Therefore, our results demonstrate that the addition of these ILs to the heme proteins decreases their thermal stability allowing the protein to be in an unfolded state at lower temperatures. Further, we describe the molecular-structural interaction of the heme proteins with the ILs (molecule like a ligand) by the PatchDocking method.

Variation in the structural changes of myoglobin in the presence of several protic ionic liquid

Protein stability in ionic solution depends on the delicate balance between protein-ion and ion-ion interactions. To address the ion specific effects on the protein, we have examined the stability of myoglobin (Mb) in the presence of buffer and ammonium-based ionic liquids (ILs) (50%, v/v). Here, fluorescence and circular dichroism (CD) spectroscopy experiments are used to study the influence of ILs on structure and stability of Mb. Our experimental results reveal that more viscous ILs (sulphate or phosphate ions) are stabilizers and therefore more biocompatible for Mb structure. Surprisingly, the less viscous ILs such as acetate anion based ILs are destabilizers for the native structure of Mb. Our results explicitly elucidate that anion variation has significant influence on Mb stability efficiency than cation variation. This study provides insight into anion effects on protein stability and explains that the intrasolvent interactions can be leveraged to enhance the stability.

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.

Does 1-Allyl-3-methylimidazolium chloride Act as a Biocompatible Solvent for Stem Bromelain?

The broader scope of ILs in chemical sciences particularly in pharmaceutical, bioanalytical and many more applications is increasing day by day. Hitherto, a very less amount of research is available in the depiction of conformational stability, activity, and thermal stability of enzymes in the presence of ILs. In the present study, the perturbation in the structure, stability, and activity of stem bromelain (BM) has been observed in the presence of 1-allyl-3-methylimidazolium chloride ([Amim][Cl]) using various techniques. This is the first report in which the influence of [Amim][Cl] has been studied on the enzyme BM. Fluorescence spectroscopy has been utilized to map out the changes in the environment around tryptophan (Trp) residues of BM and also to discuss the variations in the thermal stability of BM as an outcome of its interaction with the IL at different concentrations. Further, the work delineates the denaturing effect of high concentration of IL on enzyme structure and activity. It dictates the fact that low concentrations (0.01-0.10 M) of [Amim][Cl] are only changing the structural arrangement of the protein without having harsh consequences on its activity and stability. However, high concentrations of IL proved to be totally devastating for both activity and stability of BM. The observed decrease in the stability of BM at high concentration may be due to the combined effect of cation and anion interactions with the protein residues. The present work is successful in dictating the probable mechanism of interaction between BM and [Amim][Cl]. These results can prove to be fruitful in the studies of enzymes in aqueous IL systems since the used IL is thermally stable and nonvolatile in nature thereby providing a pathway of alteration in the activity of enzymes in potentially green systems.

Undesirable impact on structure and stability of insulin on addition of (+)-catechin hydrate with sugar

Insulin (In) based formulation has been used over decades for the cure of In-dependent diabetic patients, however, more attempts are still required to improve the remedial use of In. In this regard, the use of green tea has become widespread nowadays. However, it is unknown that (+)-catechin hydrate (CAT), a major component of green tea which enhances anti-diabetic activity of In, will or will not enhance the structure and stability of In if ingested with sugars. Interestingly, by using biophysical techniques, present study reveals the fact that the use of sugar during the intake of green tea extract may produce unwanted effects on In which may further lead to some disorders associated with In stability and also create obstacle in successful implications of In formulations.

Effect of Imidazolium-Based Ionic Liquids on the Structure and Stability of Stem Bromelain: Concentration and Alkyl Chain Length Effect

In the present work, changes in the structure and stability of stem bromelain (BM) are observed in the presence of a set of four imidazolium-based ionic liquids (ILs) 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]), using various biophysical techniques. Fluorescence spectroscopy is used to observe the changes taking place in the microenvironment around the tryptophan (Trp) residues of BM and its thermal stability because of its interactions with the ILs at different concentrations. Near-UV circular dichroism results showed that the native structure of BM remained preserved only at lower concentrations of ILs. In agreement with these results, dynamic light scattering revealed the formation of large aggregates of BM at higher concentrations of ILs, indicating the unfolding of BM. In addition to this, the results also show that higher alkyl chain length imidazolium-based ILs have a more denaturing effect on the BM structure as compared to the lower alkyl chain length ILs because of the increased hydrophobic interaction between the ILs and the BM structure. Interestingly, it is noted that low concentrations (0.01-0.10 M) of short alkyl chain ILs only alter the structural arrangement of the protein without any significant effect on its stability. However, high concentrations of all five ILs are found to disrupt the structural stability of BM.

The Role of Ionic Liquids in Protein Folding/Unfolding Studies

Ionic liquids (ILs) have emerged as novel solvent medium for several biotechnological processes in vitro. The use of ILs starts from protein extraction to catalysis to folding/ unfolding studies. ILs are becoming the most favorite non-aqueous medium for protein studies due to their unique ionic combinations (cation + anion) and tunable physical properties. In this context, several research results have been published that use of pure or aqueous IL solutions as stabilizer for proteins. Hence, herein, in this chapter, we present a collection of research work that focuses on the importance of ILs (and their mixture) in protein stabilities. In addition, we have also reviewed the unique properties of ILs as counteracting solvents for cold-induced denaturation and also their refolding properties. This report will definitely generate a new understanding for the ILs, their importance and applicability in protein folding studies.