Mechanism and Dynamics of Long-Term Stability of Cytochrome c Conferred by Long-Chain Imidazolium Ionic Liquids at Low Concentration

Abstract

Protein stability has been a concern for researchers for a long time as they are sensitive toward their environment. Mostly proteins during experiments require medium that keep them stable at room temperature (RT). Recent research utilizing ionic liquids (ILs) to stabilize protein has gained much importance. Although a few ILs have been claimed to suit this requirement, reported studies employ IL concentrations that might produce irreversible denaturation and aggregation. This study demonstrates the first report for long-term stabilization of horse heart cytochrome c (h-cyt c) by long-chain imidazolium ILs at far low concentration (1 mM) of IL when stored at RT. Long-chain imidazolium ILs until now were less familiar for their stabilizing nature toward protein. A significant increase in the helical content of h-cyt c (dissolved state) was observed with prolonged structural stability (secondary and tertiary) for about 6 months in aqueous solutions of 1-methyl-3-octyl imidazolium chloride [C8mim]­[Cl] and 1-decyl-3-methylimidazolium chloride [C10mim]­[Cl]. The in-depth mechanism discussed suggests interaction of ILs with amino acid residues of h-cyt c, which rigidifies the loop regions with reduced mobility; hence, prolonged stability is achieved. The study firmly advocates the use of long-chain imidazolium ILs as the potent inhibitor against denaturation during storage of h-cyt c at RT.

Implication of Threonine‐Based Ionic Liquids on the Structural Stability, Binding and Activity of Cytochrome c

“…Addition of both [Ch][Thr] and [Thr][NO 3 ] ILs (up to 10 mM) to the Cyt c did not change the spectral characteristics that confirmed tertiary structure stability of Cyt c in presence of both ILs. Patel and coworkers [18a] in their study explained that the ILs bearing positive charge and hydrophobic long alkyl chains interact significantly with the hydrophobic amino acid (side chain) residues in backbone of Cyt c. This interaction results in stabilization of the tertiary structure and at the same time the heme core also remains intact. When [Ch][Thr] IL was added to Cyt c, the positive charge of choline cation and hydrophobic alkyl chains in threonine anion stabilize the Cyt c tertiary structure.…”

“…Based on the explanation of Singh et al., [18a] the longer lifetime enhancement observed here by increasing [Ch][Thr] IL concentration signifies the compactness of Cyt c structure due to the restriction in the movement of its loops (hydrophobicity around tryptophan increases) which increases the long duration structural stability of Cyt c. But in [Thr][NO 3 ] IL (see Table 1) where threonine is cation, it exhibited opposite trend i. e. reduction in both longer lifetime (τ 3 ) and average lifetime (τ av ) was observed which could be the consequence of polarity change. The reduction in τ 3 value indicated that with the addition of [Thr][NO 3 ] IL to Cyt c, the polarity around Trp59 region increased followed by conformational changes that started the Cyt c protein denaturation at higher IL concentration [18a,28] . Similar observations were noticed while analyzing CD and UV‐Vis spectroscopic results.…”

Probing the Intercalation of Noscapine from Sodium Dodecyl Sulfate Micelles to Calf Thymus Deoxyribose Nucleic Acid: A Mechanistic Approach

“…Time-resolved fluorescence decay is a very sensitive tool for studying the nature of hydration as well as the relaxation dynamics of the drug in aqueous and micellar environments and also the excited state interactions of the probe. 39,45−47 This gives the information about the residence of the probe within different micro heterogeneous mediums. Herein, we observe the fluorescence lifetime of NOS in different environments to find out its replacement from micelles to the ctDNA in the composite medium.…”

Use of Ionic Liquids in Protein and DNA Chemistry

“…However, a change in the tertiary structure of protein was also reported at higher concentrations of ionic liquids (Byrne et al, 2007 ; Bihari et al, 2010 ). Singh et al showed that long-chain imidazolium-based ionic liquids stabilize the horse heart cytochrome c (h-cyt-c) for a long term and at very low concentrations (1mM) (Singh et al, 2018 ). However, the presence of water in ionic liquid-protein mixtures is shown to have a distinct effect on the structural features.…”