Pannuru Venkatesu

Influence of Osmolytes and Denaturants on the Structure and Enzyme Activity of α-Chymotrypsin

Enzymes are very sensitive and highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of cosolvents, the fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways, and alternatively, enzymes can be stabilized or destabilized. To understand the contribution of osmolytes and denaturants on the stabilization, related to the associated structural changes and enzyme activity of alpha-chymotrypsin (CT), we have monitored differential scanning calorimeter (DSC), circular dichroism (CD), enzyme activity, and gel electrophoresis as a function of osmolyte or denaturant concentration. The present investigation compares the compatibility of osmolytes and deleterious effects of denaturants on the structure, function, and enzyme activity of CT. This comparison has provided new important insight on the contribution of cosolvent effects on protein folding/unfolding, enzyme activity, and understanding of protein-solvent interactions. Our DSC results reveal that the enthalpy change (DeltaH) and Gibbs free energy of change (DeltaG(u)) of CT in osmolyte (trimethylamine N-oxide (TMAO), betaine, sarcosine, proline, and sucrose) increase linearly as osmolyte concentration increases, while those values decrease sharply in the presence of denaturants (urea and guanidine hydrochloride (GdnHCl)). The modifications in the secondary structure of this beta/beta protein, as quantified by the CD spectra, showed reasonable enhancement for beta-strands in the presence of the osmolytes as compared to buffer, which contributes to its stabilization power. Evidently, we observed that naturally occurring osmolytes have a dominant contribution to the stabilization of CT while not enhancing its enzyme activity. In contrast, our results revealed that the denaturants enhanced the surface of the enzyme by binding to the surface of CT, which leads to zero enzyme activity.

Measurements and Molecular Interactions for N,N-Dimethylformamide with Ionic Liquid Mixed Solvents

To understand the molecular interactions between N,N-dimethylformamide (DMF) with two families of ionic liquids (ILs), we have measured thermophysical properties such as densities (rho) and ultrasonic sound velocities (u) over the whole composition range at 25 degrees C under atmospheric pressure. The excess molar volume (V(E)) and the deviation in isentropic compressibilities (DeltaK(s)) were predicted using these properties as a function of the concentration of IL. These results are fitted to the Redlich-Kister polynomials. The materials investigated in the present study included two families of ILs such as ammonium salts and imidazolium salts. Diethylammonium acetate ([Et(2)NH][CH(3)COO], DEAA), triethylammonium actetate ([Et(3)NH][CH(3)COO], TEAA), triethylammonium dihydrogen phosphate ([Et(3)NH][H(2)PO(4)], TEAP), and triethylammonium sulfate ([Et(3)NH][HSO(4)], TEAS) are ammonium salts and 1-benzyl-3-methylimidazolium chloride ([Bmim][Cl]) belongs to the imidazolium family. The intermolecular interactions and structural effects were analyzed on the basis of the measured and the derived properties. A qualitative analysis of the results is discussed in terms of the ion-dipole, ion-pair interactions, and hydrogen bonding between ILs and DMF molecules and their structural factors.

Ionic Liquid Modifies the Lower Critical Solution Temperature (LCST) of Poly(N-isopropylacrylamide) in Aqueous Solution

The effect of the imidazolium based ionic liquid (IL) 1-benzyl-3-methylimidazolium tetrafluoroborate ([Bzmim][BF(4)]) was investigated on the lower critical solution temperature (LCST) of poly(N-isopropylacrylamide) (PNIPAM) in aqueous solution by using fluorescence, viscometric, and dynamic light scattering (DLS) techniques. The measurements were performed at four different [Bmim][BF(4)] concentrations (1-4 mg/mL) in PNIPAM aqueous solution. Our experimental results elucidate that the IL induces the collapsed globular structure of polymer, facilitated by the weakening of hydrogen bonds between the amide group of the polymer and water molecules; therefore, IL destabilizes the hydrated macromolecule structure. We observed that the phase transition of PNIPAM aqueous solution abruptly shifts down with increasing IL concentration mainly due to hydrophobic collapse and aggregation of a macromolecule. These results unambiguously reveal that the imidazolium based IL significantly affected the phase transition of PNIPAM and ruptured the hydrogen bonding between polymer and water molecules, and eventually the hydrated macromolecule structure was destabilized.

A protic ionic liquid attenuates the deleterious actions of urea on α-chymotrypsin

We present a biocompatible ionic liquid, triethyl ammonium acetate (TEAA), that attenuates the denaturation action of a non-ionic chaotrope, urea, on the industrially relevant proteolytic enzyme α-chymotrypsin.

Temperature effect on the molecular interactions between ammonium ionic liquids and N,N-dimethylformamide.

In view of the wide scope of molecular interactions between the highly polar compound of N,N-dimethylformamide (DMF) and ammonium ionic liquids (ILs), we have measured thermophysical properties such as densities (ρ) and ultrasonic sound velocities (u) over the whole composition range at temperatures ranging from 25 to 50 °C under atmospheric pressure. To gain some insight into the several aggregations of molecular interactions present in these mixed solvents, we predicted the excess molar volume (V(E)) and the deviations in isentropic compressibilities (ΔK(s)) as a function of the concentration of IL. These results are fitted to the Redlich-Kister polynomials. The materials investigated in the present study included the hydroxide series of ammonium ILs of tetramethylammonium hydroxide [(CH(3))(4)N][OH] (TMAH), tetraethylammonium hydroxide [(C(2)H(5))(4)N][OH] (TEAH), and tetrapropylammonium hydroxide [(C(2)H(7))(4)N][OH] (TPAH). The intermolecular interactions and structural effects were analyzed on the basis of the measured and the derived properties. A qualitative analysis of the results is discussed in terms of the ion-dipole and ion-pair interactions, and hydrogen bonding between ILs and DMF molecules and their structural factors.

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.

Temperature dependence measurements and structural characterization of trimethyl ammonium ionic liquids with a highly polar solvent.

We report the synthesis and characterization of a series of an ammonium ionic liquids (ILs) containing acetate, dihydrogen phosphate, and hydrogen sulfate anions with a common cation. To characterize the thermophysical properties of these newly synthesized ILs with the highly polar solvent N,N-dimethylformamide (DMF), precise measurements such as densities (ρ) and ultrasonic sound velocities (u) over the whole composition range have been performed at atmospheric pressure and over wide temperature ranges (25-50 °C). The excess molar volume (V(E)) and the deviation in isentropic compressibilities (Δκ(s)) were predicted using these temperature dependence properties as a function of the concentration of ILs. The Redlich-Kister polynomial was used to correlate the results. The ILs investigated in the present study included trimethylammonium acetate [(CH(3))(3)NH][CH(3)COO] (TMAA), trimethylammonium dihydrogen phosphate [(CH(3))(3)NH][H(2)PO(4)] (TMAP), and trimethylammonium hydrogen sulfate [(CH(3))(3)NH][HSO(4)] (TMAS). The intermolecular interactions and structural effects were analyzed on the basis of the measured and the derived properties. In addition, the hydrogen bonding between ILs and DMF has been demonstrated using semiempirical calculations with help of Hyperchem 7. A qualitative analysis of the results is discussed in terms of the ion-dipole, ion-pair interactions, and hydrogen bonding between ILs and DMF molecules and their structural factors. The influence of the anion of the protic IL, namely, acetate (CH(3)COO), dihydrogen phosphate (H(2)PO(4)), and hydrogen sulfate (HSO(4)), on the thermophysical properties is also provided.

Influence of alkyl chain length and temperature on thermophysical properties of ammonium-based ionic liquids with molecular solvent.

Mixing of ionic liquids (ILs) with molecular solvent can expand the range of structural properties and the scope of molecular interactions between the molecules of the solvents. Exploiting of these phenomena essentially require a basic fundamental understanding of mixing behavior of ILs with molecular solvents. In this context, a series of protic ILs possessing tetra-alkyl ammonium cation [R(4)N](+) with commonly used anion hydroxide [OH](-) were synthesized and characterized by temperature dependent thermophysical properties. The ILs [R(4)N](+)[OH](-) are varying only in the length of alkyl chain (R is methyl, ethyl, propyl, or butyl) of tetra-alkyl ammonium on the cationic part. The ILs used for the present study included tetramethyl ammonium hydroxide [(CH(3))(4)N](+)[OH](-) (TMAH), tetraethyl ammonium hydroxide [(C(2)H(5))(4)N](+)[OH](-) (TEAH), tetrapropyl ammonium hydroxide [(C(3)H(7))(4)N](+)[OH](-) (TPAH) and tetrabutyl ammonium hydroxide [(C(4)H(9))(4)N](+)[OH](-) (TBAH). The alkyl chain length effect has been analyzed by precise measurements such as densities (ρ), ultrasonic sound velocity (u), and viscosity (η) of these ILs with polar solvent, N-methyl-2-pyrrolidone (NMP), over the full composition range as a function of temperature. The excess molar volume (V(E)), the deviation in isentropic compressibility (Δκ(s)) and deviation in viscosity (Δη) were predicted using these properties as a function of the concentration of ILs. Redlich-Kister polynomial was used to correlate the results. A qualitative analysis of the results is discussed in terms of the ion-dipole, ion-pair interactions, and hydrogen bonding between ILs and NMP molecules. Later, the hydrogen bonding features between ILs and NMP were also analyzed using a molecular modeling program with the help of HyperChem 7.

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.

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.