Determining the structure of a protein and its transformation under different conditions is key to understanding its activity. The structural stability and activity of proteins in aqueous-organic solvent mixtures, which is an intriguing topic of research in biochemistry, is dependent on the nature of the protein and the properties of the medium. Herein, the effect of a commonly used cosolvent, dimethyl sulfoxide (DMSO), on the structure and conformational dynamics of bovine serum albumin (BSA) protein is studied by fluorescence correlation spectroscopy (FCS) measurements on fluorescein isothiocyanate (FITC)-labeled BSA. The FCS study reveals a change of the hydrodynamic radius of BSA from 3.7 nm in the native state to 7.0 nm in the presence of 40% DMSO, which suggests complete unfolding of the protein under these conditions. Fluorescence self-quenching of FITC has been exploited to understand the conformational dynamics of BSA. The time constant of the conformational dynamics of BSA is found to change from 35 μs in its native state to 50 μs as the protein unfolds with increasing DMSO concentration. The FCS results are corroborated by the near-UV circular dichroism spectra of the protein, which suggest a loss of its tertiary structure with increasing concentration of DMSO. The intrinsic fluorescence of BSA and the fluorescence response of 1-anilinonaphthalene-8-sulfonic acid, used as a probe molecule, provide information that is consistent with the FCS measurements, except that aggregation of BSA is observed in the presence of 40% DMSO in the ensemble measurements.

Choline dioctylsulfosuccinate [Cho][AOT] (a surface active ionic liquid) has been found to induce all-α to α + β conformational transition in the secondary structure of enzyme cytochrome c (Cyt c) with an enhanced peroxidase activity in its aqueous vesicular phase at pH 7.0. [Cho][AOT] interacted with Cyt c distinctly at three critical concentrations (aggregation C1, saturation C2 and vesicular C3) as detected from isothermal titration calorimetric analysis. Oxidation of heme iron was observed from the disappearance of the Q band in the UV-vis spectra of Cyt c upon [Cho][AOT] binding above C3. Circular dichroism analysis (CD) has shown the loss in both the secondary (190-240 nm) and tertiary (250-300 nm) structure of Cyt c in the monomeric regime until C1, followed by their stabilization until the pre-vesicular regime (C1 → C3). Loss in both the secondary and tertiary structure has been observed in the post-vesicular regime with the change in Cyt c conformation from all-α to α + β which is similar to the conformational changes of Cyt c upon binding with mitochondrial membrane (Biochemistry 1998, 37, 6402-6409), thus citing the potential utility of [Cho][AOT] membranes as an artificial analog for in vitro bio-mimicking. Fluorescence correlation spectroscopy (FCS) measurements confirm the unfolding of Cyt c in the vesicular phase. Dynamic light scattering experiments have shown the contraction of [Cho][AOT] vesicles upon Cyt c binding driven by electrostatic interactions observed by charge neutralization from zeta potential measurements. [Cho][AOT] has been found to enhance the peroxidase activity of Cyt c with maximum activity at C3, observed using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt as the substrate in the presence of hydrogen peroxide. This result shows the relevance of tuning ionic liquids to surfactants for bio-mimicking of specific membrane protein-lipid interactions.

Ability of the ionic liquids to alter the structural stability of proteins in aqueous solution is a topic of considerable interest in modern bioscientific research because of possible applications of these substances in protein purification and as refolding agents. A few early studies involving the imidazolium ionic liquids have demonstrated their role as both denaturants and refolding agents. As the influence of an ionic liquid on a given protein depends on the identity of both species, it is necessary to extend the studies to a wider number of ionic liquids and proteins to obtain insight into the mechanism of interaction between the two and to arrive at a comprehensive picture. It is in this context that we have studied the effect of two morpholinium salts, [Mor1,2][Br] and [Mor1,4][Br], differing in the alkyl chain length of cation, on chicken egg white lysozyme in its native and chemically denatured states employing primarily the fluorescence correlation spectroscopy (FCS) technique. Fluorescence signal of Alexa488-labeled lysozyme (A488-Lysz) has been used to determine the changes in hydrodynamic radius of protein in the presence of additives. The results reveal a conformational dynamics of lysozyme with a time constant of 56 ± 10 μs in its native state. It is observed, when in its native state, both the morpholinium salts induce structural changes of lysozyme. However, when in its unfolded state, [Mor1,4][Br] at low concentration compacts the protein, but at higher concentration, it stabilizes the unfolded state, unlike [Mor1,2][Br], which compacts lysozyme at both low and high concentrations. A comparison of the effect of these salts and arginine, a protein stabilizer, on lysozyme indicates that [Mor1,2][Br] is a superior compacting agent for the unfolded state of the protein compared to arginine.

The structural integrity of a nucleic acid under various conditions determines its utility in biocatalysis and biotechnology. Exploration of the ionic liquids (ILs) for extraction of DNA and other nucleic acid based applications requires an understanding of the nature of interaction between the IL and DNA. Considering these aspects, we have studied the interaction between calf-thymus DNA and a less toxic morpholinium IL, [Mor1,2][Br], employing fluorescence correlation spectroscopy (FCS), conventional steady state and time-resolved fluorescence, circular dichroism (CD) and molecular docking techniques. While the CD spectra indicate the stability of DNA and retention of its B-form in the presence of the morpholinium IL, the docking study reveals that [Mor1,2](+) binds to the minor groove of DNA with a binding energy of -4.57 kcal mol(-1). The groove binding of the cationic component of the IL is corroborated by the steady state fluorescence data, which indicated displacement of a known minor groove binder, DAPI, from its DNA-bound state on addition of [Mor1,2][Br]. The FCS measurements show that the hydrodynamic radius of DNA remains more or less constant in the presence of [Mor1,2][Br], thus suggesting that the structure of DNA is retained in the presence of the IL. DNA melting experiments show that the thermal stability of DNA is enhanced in the presence of morpholinium IL.

Photophysical properties of a natural plant alkaloid, ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole), which comprises both proton donating and accepting sites, have been studied in different solvents using steady state and time-resolved fluorescence techniques primarily to understand the origin of dual fluorescence that this molecule exhibits in some specific alcoholic solvents. Ground and excited state calculations based on density functional theory have also been carried out to help interpretation of the experimental data. It is shown that the long-wavelength emission of the molecule is dependent on the hydrogen bond donating ability of the solvent, and in methanol, this emission band arises solely from an excited state reaction. However, in ethylene glycol, both ground and excited state reactions contribute to the long wavelength emission. The time-resolved fluorescence data of the system in methanol and ethylene glycol indicates the presence of two different hydrogen bonded species of ellipticine of which only one participates in the excited state reaction. The rate constant of the excited state reaction in these solvents is estimated to be around 4.2-8.0 × 10(8) s(-1). It appears that the present results are better understood in terms of solvent-mediated excited state intramolecular proton transfer reaction from the pyrrole nitrogen to the pyridine nitrogen leading to the formation of the tautomeric form of the molecule rather than excited state proton transfer from the solvents leading to the formation of the protonated form of ellipticine.