Upendra Kumar Singh

Spectroscopic and docking studies on the interaction between pyrrolidinium based ionic liquid and bovine serum albumin

The interaction of synthesized ionic liquid, 1-butyl-1-methyl-2-oxopyrrolidinium bromide (BMOP) and bovine serum albumin (BSA) was investigated using UV-Vis, FT-IR, steady state and time resolved fluorescence measurements and docking studies. Steady state spectra revealed that BMOP strongly quenched the intrinsic fluorescence of BSA through dynamic quenching mechanism. The corresponding thermodynamic parameters; Gibbs free energy change (ΔG), entropy change (ΔS) and enthalpy change (ΔH) showed that the binding process was spontaneous and entropy driven. It is also indicated that hydrophobic forces play a key role in the binding of BMOP to BSA. The synchronous fluorescence spectroscopy reveals that the conformation of BSA changed in the presence of BMOP. The shift in amide I band of FT-IR spectrum of BSA suggested unfolding of the protein secondary structure upon the addition of BMOP. In addition, the molecular modeling study of BSA-BMOP system shows that BMOP binds with BSA at the interface between two sub domains IIA and IIIA, which is located just above the entrance of the binding pocket of IIA through hydrophobic and hydrogen bond interactions in which hydrophobic interaction are dominated.

Molecular investigation of the interaction between ionic liquid type gemini surfactant and lysozyme: A spectroscopic and computational approach

Herein, we are reporting the interaction of ionic liquid type gemini surfactant, 1,4‐bis(3‐dodecylimidazolium‐1‐yl) butane bromide ([C12−4‐C12im]Br2) with lysozyme by using Steady state fluorescence, UV‐visible, Time resolved fluorescence, Fourier transform‐infrared (FT‐IR) spectroscopy techniques in combination with molecular modeling and docking method. The steady state fluorescence spectra suggested that the fluorescence of lysozyme was quenched by [C12−4‐C12im]Br2 through static quenching mechanism as confirmed by time resolved fluorescence spectroscopy. The binding constant for lysozyme‐[C12−4‐C12im]Br2 interaction have been measured by UV‐visible spectroscopy and found to be 2.541 × 105M−1. The FT‐IR results show conformational changes in the secondary structure of lysozyme by the addition of [C12−4‐C12im]Br2. Moreover, the molecular docking study suggested that hydrogen bonding and hydrophobic interactions play a key role in the protein‐surfactant binding. Additionally, the molecular dynamic simulation results revealed that the lysozyme‐[C12−4‐C12im]Br2 complex reaches an equilibrium state at around 3 ns.

Spectroscopic and molecular modelling analysis of the interaction between ethane-1,2-diyl bis(N,N-dimethyl-N-hexadecylammoniumacetoxy)dichloride and bovine serum albumin.

Several spectroscopic approaches namely fluorescence, time-resolved fluorescence, UV-visible, and Fourier transform infra-red (FT-IR) spectroscopy were employed to examine the interaction between ethane-1,2-diyl bis(N,N-dimethyl-N-hexadecylammoniumacetoxy)dichloride (16-E2-16) and bovine serum albumin (BSA). Fluorescence studies revealed that 16-E2-16 quenched the BSA fluorescence through a static quenching mechanism, which was further confirmed by UV-visible and time-resolved fluorescence spectroscopy. In addition, the binding constant and the number of binding sites were also calculated. The thermodynamic parameters at different temperatures (298 K, 303 K, 308 K and 313 K) indicated that 16-E2-16 binding to BSA is entropy driven and that the major driving forces are electrostatic interactions. Decrease of the α-helix from 53.90 to 46.20% with an increase in random structure from 22.56 to 30.61% were also observed by FT-IR. Furthermore, the molecular docking results revealed that 16-E2-16 binds predominantly by electrostatic and hydrophobic forces to some residues in the BSA sub-domains IIA and IIIA.

Effect of pyrrolidinium based ionic liquid on the channel form of gramicidin in lipid vesicles

The present work is focused on the interaction between membrane bound gramicidin and 1-butyl-1-methyl-2-oxopyrrolidinium bromide (BMOP) ionic liquid. Ionic liquids (ILs) are solvents that are often liquid at room temperature and composed of organic cation and appropriate anion. The gramicidin peptide forms prototypical ion channels for cations, which have been extensively used to study the organization, dynamics, and function of membrane spanning channels. The interaction was studied by circular dichroism, steady state, time-resolved fluorescence spectroscopy in combination with dynamic surface tension and field emission scanning electron microscopic methods (FESEM). The results obtained from circular dichroism shows that the BMOP interacts with the channel form of gramicidin in lipid vesicle without any considerable effect on its conformation. The Red-edge excitation shift (REES) also supported the above findings. In addition, the fluorescence studies suggested that BMOP makes ground state complex with ion channel, which was further supported by time resolved measurements. Furthermore, dynamic surface tension analysis shows the faster adsorption of BMOP with membrane bound gramicidin at the air-water interface. Additionally, FESEM results indicated that BMOP forms a film around the membrane bound gramicidin at higher concentration. These results are potentially useful to analyze the effect of ionic liquids on the behaviour of membrane proteins.

Refolding of urea denatured cytochrome c: Role of hydrophobic tail of the cationic gemini surfactants

The refolding of urea denatured horse heart cytochrome c (h-cyt-c) under the influence of ester based cationic gemini surfactants [ethane-1, 2-diyl bis(N, N-dimethyl-N-alkylammoniumacetoxy) dichlorides] 16-E2-16, 14-E2-14 and 12-E2-12 (n-E2-n) was performed by using UV-visible, fluorescence and circular dichroism (CD) spectroscopic techniques. We found that n-E2-n geminis promote the formation of molten globule (MG) like state upon addition into the urea denatured h-cyt-c. The comparative study of refolding of denatured h-cyt-c with n-E2-n, cationic gemini surfactant show stabilization of MG-like state influenced by hydrophobic interactions. The formation of MG-like state from the unfolded protein confirms the presence of some regular structures induced by n-E2-n gemini surfactants. Thermodynamic parameters for refolding of h-cyt-c by n-E2-n were also measured and the m-values of all the refolded states of h-cyt-c by n-E2-n show marked difference. The higher m-values correspond to the larger hydrophobic chain length indicates that refolding ability of the n-E2-n depends on the alkyl chain length. The result is related to the stronger hydrophobic forces due to the presence of two head groups and two hydrophobic hydrocarbon tails. This study showed that these cationic gemini surfactants were efficiently utilized in the protein refolding studies.

Comparative effect of cationic gemini surfactant and its monomeric counterpart on the conformational stability and activity of lysozyme

Protein interactions with surfactants are dependent on their physiochemical properties. The effect of cationic gemini surfactant hexanediyl-α,ω-bis-(N-(2-hydroxyethyl)-N-methylhexadecylammonium dibromide) on the stability and activity of hen egg white lysozyme was compared with its monomeric counterpart N-(2-hyroxyethyl)-N,N-dimethylhexadecylammonium bromide at pre and post micellar concentrations. This study utilizes circular dichroism (CD), steady-state fluorescence spectroscopy, extrinsic fluorescence spectroscopy, time-resolved fluorescence spectroscopy, UV-visible spectroscopy, molecular docking and turbidity assays to resolve the conformational stability and antibacterial activity of lysozyme in the presence of surfactants. Micelles of both cationic surfactants were observed to stabilize the conformation of the protein, however, gemini was found to stabilize it in a much higher micellar concentration range. Detailed analysis of the time-resolved fluorescence spectroscopy results suggests contribution of the lifetime values of Trp62 and Trp108 to the overall conformation change of lysozyme with the increase in concentration of the respective surfactants, which is further correlated with the steady-state fluorescence and CD spectroscopy results. Furthermore, from the CD analysis it was found that the cationic single chain surfactant strongly perturbs the secondary and tertiary structure of the protein as compared to the gemini surfactant. Through docking results, it was found that the gemini surfactant binds weakly with lysozyme as compared to the single chain surfactant. Specifically, the antibacterial activity of lysozyme was found to be increased in the presence of cationic gemini surfactant, which extrapolates the use of these surfactants in pharmaceutics and industries.

Dynamics of Ionic Liquid-Assisted Refolding of Denatured Cytochrome c: A Study of Preferential Interactions toward Renaturation

In vitro refolding of denatured protein and the influence of the alkyl chain on the refolding of a protein were tested using long chain imidazolium chloride salts, 1-methyl-3-octylimidazolium chloride [C8mim][Cl], and 1-decyl-3-methylimidazolium chloride [C10mim][Cl]. The horse heart cytochrome c (h-cyt c) was denatured by urea and guanidinium hydrochloride (GdnHCl), as well as by base-induced denaturation at pH 13, to provide a broad overview of the overall refolding behavior. The variation in the alkyl chain of the ionic liquids (ILs) showed a profound effect on the refolding of denatured h-cyt c. The ligand-induced refolding was correlated to understand the mechanism of the conformational stability of proteins in aqueous solutions of ILs. The results showed that the long chain ILs having the [C8mim]+ and [C10mim]+ cations promote the refolding of alkali-denatured h-cyt c. The IL having the [C10mim]+ cation efficiently refolded the alkali-denatured h-cyt c with the formation of the MG state, whereas the IL having the [C8mim]+ cation, which is known to be compatible for protein stability, shows slight refolding and forms a different transition state. The lifetime results show successful refolding of alkaline-denatured h-cyt c by both of the ILs, however, more refolding was observed in the case of [C10mim][Cl], and this was correlated with the fast and medium lifetimes (τ1 and τ2) obtained, which show an increase accompanied by an increase in secondary structure. The hydrophobic interactions plays an important role in the refolding of chemically and alkali-denatured h-cyt c by long chain imidazolium ILs. The formation of the MG state by [C10mim][Cl] was also confirmed, as some regular structure exists far below the CMC of IL. The overall results suggested that the [C10mim]+ cation bound to the unfolded h-cyt c triggers its refolding by electrostatic and hydrophobic interactions that stabilize the MG state.

Effect of bovine serum albumin on the surface properties of ionic liquid-type Gemini surfactant

ABSTRACT The effect of bovine serum albumin on the surface properties of IL-type gemini surfactant ([C10-4-C10im]Br2), have been investigated by surface tension method. The critical micelle concentration (CMC) as a function of BSA concentrations at various temperatures was investigated. The CMC of [C10-4-C10im]Br2 increases with increasing the concentration of BSA as well as the temperature of the system. The interfacial parameters viz; maximum surface excess concentration (Γmax), the minimum area per molecule (Amin), and surface pressure at CMC (Πcmc) were calculated. In addition, thermodynamic parameters of adsorption and micellization were evaluated by using surface tension data. The results indicated that the binding of [C10-4-C10im]Br2 to BSA is spontaneous and exothermic in nature. The process is entropy driven and hydrophobic interactions are the major driving forces. GRAPHICAL ABSTRACT