Effect of ethanol on the size and structure of a protein cytochrome C (Cyt C) is investigated using fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations. For FCS studies, Cyt C is covalently labeled with a fluorescent probe, alexa 488. FCS studies indicate that on addition of ethanol, the size of the protein varies non-monotonically. The size of Cyt C increases (i.e., the protein unfolds) on addition of alcohol (ethanol) up to a mole fraction of 0.2 (44.75% v/v) and decreases at higher alcohol concentration. In order to provide a molecular origin of this structural transition, we explore the conformational free energy landscape of Cyt C as a function of radius of gyration (R) at different compositions of water-ethanol binary mixture using MD simulations. Cyt C exhibits a minimum at R ∼ 13 Å in bulk water (0% alcohol). Upon increasing ethanol concentration, a second minimum appears in the free energy surface with gradually larger R up to χ ∼ 0.2 (44.75% v/v). This suggests gradual unfolding of the protein. At a higher concentration of alcohol (χ > 0.2), the minimum at large R vanishes, indicating compaction. Analysis of the contact map and the solvent organization around protein indicates a preferential solvation of the hydrophobic residues by ethanol up to χ = 0.2 (44.75% v/v) and this causes the gradual unfolding of the protein. At high concentration (χ = 0.3 (58% v/v)), due to structural organization in bulk water-ethanol binary mixture, the extent of preferential solvation by ethanol decreases. This causes a structural transition of Cyt C towards a more compact state.
Aqueous binary mixtures have received immense attention in recent years because of their extensive application in several biological and industrial processes. The water-ethanol binary mixture serves as a unique system because it exhibits composition-dependent alteration of dynamic and thermodynamic properties. Our present work demonstrates how different compositions of water-ethanol binary mixtures affect the pair hydrophobicity of different hydrophobes. Pair hydrophobicity is measured by the depth of the first minimum (contact minima) of potential of mean force (PMF) profile between two hydrophobes. The pair hydrophobicity is found to be increased with addition of ethanol to water up to a mole fraction of x = 0.10 and decreased with further addition of ethanol. This observation is shown to be true for three (methane-methane, isobutane-isobutane, and toluene-toluene) different pairs of hydrophobes. Decomposition of PMF into enthalpic and entropic contribution indicates a switch from entropic to enthalpic stabilization of the contact minimum upon addition of ethanol to water. The gain in mixing enthalpy of the binary solvent system upon association of two hydrophobes is found to be the determining factor for the stabilization of contact minimum. Several static/dynamics quantities (average composition fluctuations, diffusion coefficients, fluctuations in total dipole moment, propensity of ethyl-ethyl association, etc) of the ethanol-water binary mixture also show irregularities around x = 0.10-0.15. We have also discovered that the hydrogen bonding pattern of ethanol rather than water reveals a change in trend near the similar composition range. As the anomalous behavior of the physical/dynamical properties along with the pair hydrophobicity in an aqueous binary mixture of amphiphilic solutes is a common phenomenon, our results may provide a general viewpoint on these aspects.
In this review, we give a brief overview on how the interaction of proteins with ionic liquids, alcohols and dimethyl sulfoxide (DMSO) influences the stability, conformational dynamics and function of proteins/enzymes. We present experimental results obtained from fluorescence correlation spectroscopy on the effect of ionic liquid or alcohol or DMSO on the size (more precisely, the diffusion constant) and conformational dynamics of lysozyme, cytochrome c and human serum albumin in aqueous solution. The interaction of ionic liquid with biomolecules (e.g. protein, DNA etc.) has emerged as a current frontier. We demonstrate that ionic liquids are excellent stabilizers of protein and DNA and, in some cases, cause refolding of a protein already denatured by chemical denaturing agents. We show that in ethanol-water binary mixture, proteins undergo non-monotonic changes in size and dynamics with increasing ethanol content. We also discuss the effect of water-DMSO mixture on the stability of proteins. We demonstrate how large-scale molecular dynamics simulations have revealed the molecular origin of this observed phenomenon and provide a microscopic picture of the immediate environment of the biomolecules. Finally, we describe how favorable interactions of ionic liquids may be utilized for in situ generation of fluorescent gold nano-clusters for imaging a live cell.
Structural relaxation of the acridine orange (AO) dimer in bulk water and inside a single live lung cell is studied using time resolved confocal microscopy and molecular dynamics (MD) simulations. The emission maxima (λem (max)∼ 630 nm) of AO in a lung cancer cell (A549) and a non-cancer lung fibroblast cell (WI38) suggest that AO exists as a dimer inside the cell. Time-dependent red shift in emission maximum indicates dynamic relaxation of the AO dimer (in the excited state) with a time constant of 500-600 ps, both in bulk water and inside the cell. We have calculated the equilibrium relaxation dynamics of the AO dimer in the ground state using MD simulations and found a slow component of time scale ∼ 350 ps. The intra- and inter-molecular components of the total relaxation dynamics of the AO dimer reveal the presence of a slow component of the order of a few hundred picoseconds. Upon restricting intra-molecular dye dynamics by harmonic constraint between AO monomers, the slow component vanishes. Combining the experimental observations and MD simulation results, we ascribe the slow component of the dynamic relaxation of the AO dimer to the structural relaxation, namely, fluctuations in the distance between the two monomers and associated fluctuation in the number of water molecules.
The self-aggregation property of the perfluoro group containing molecules makes it important in the research fields of biology and polymer and organic synthesis. In the quest of understanding the role of the perfluoro group on the photophysical properties of perfluoro-containing molecules in biologically important fluoroethanol solvents, we have applied photophysical as well as molecular dynamics simulation techniques to explore the properties of perfluoro groups containing molecule coumarin-153 (C153) in ethanol (ETH), monofluoroethanol (MFE), difluoroethanol (DFE), and trifluoroethanol (TFE) and compared them with the molecules without perfluoro moiety, namely coumarin-6H (C6H) and coumarin-480 (C480). In contrast to C6H and C480, the excited state lifetime of C153 in fluorinated ETHs is not monotonic. The excited state lifetime of C153 decreases in MFE and DFE as compared to ETH, whereas in TFE, it increases as compared to MFE and DFE. Molecular dynamics simulation reveals that the carbon terminal away from the OH group of fluorinated ETHs has a preferential orientation near the perfluoro (CF3) group of C153. In MFE and DFE, the CF3 group of C153 prefers to have a CF2-F⋯H -(CHF) type of electrostatic interaction over CF2-F⋯F -(CH2) kind of dispersion interaction which increases the rate of nonradiative decay, probably due to the electrostatic nature of the CF2-F⋯H -(CHF) hydrogen bond. On the other hand, in TFE, C-F⋯ F-C type of dispersion interaction, also known as fluorous interaction, takes place between the CF3 groups of C153 and TFE which decreases the rate of nonradiative rate as compared to MFE and DFE, leading to the increased lifetime of C153 in TFE. Photophysical and MD simulation studies clearly depict that the structural organization of solvents and their interaction with the fluorocarbon group are crucial factors for the photophysical behavior of the fluorocarbon containing molecules.
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