Thermal Reversibility and Structural Stability in Lysozyme Induced by Epirubicin Hydrochloride

Abstract

Herein we report the binding interactions between lysozyme (Lyz) and an anthracycline drug, epirubicin hydrochloride (EPR), through an extensive spectroscopic approach at both ensemble average and single molecular resolution. Our steady-state and time-resolved fluorescence spectroscopy reveals that the drug-induced fluorescence quenching of the protein proceeds through a static quenching mechanism. Isothermal titration calorimetry (ITC) and steady-state experiments reveal almost similar thermodynamic signatures of the drug–protein interactions. The underlying force that plays pivotal roles in the said interaction is hydrophobic in nature, which is enhanced in the presence of a strong electrolyte (NaCl). Circular dichroism (CD) spectra indicate that there is a marginal increase in the secondary structure of the native protein (α-helical content increases from 26.9 to 31.4% in the presence of 100 μM EPR) upon binding with the drug. Fluorescence correlation spectroscopy (FCS) was used to monitor the changes in structure and conformational dynamics of Lyz upon interaction with EPR. The individual association (K ass = 0.33 × 106 ms–1 M–1) and dissociation (K diss = 1.79 ms–1) rate constants and the binding constant (K b = 1.84 × 105 M–1) values, obtained from fluctuations of fluorescence intensity of the EPR-bound protein, have also been estimated. AutoDock results demonstrate that the drug molecule is encapsulated within the hydrophobic pocket of the protein (in close proximity to both Trp62 and Trp108) and resides ∼20 Å apart from the covalently labelled CPM dye. Förster resonance energy transfer (FRET) studies proved that the distance between the donor (CPM) and the acceptor (EPR) is ∼22 Å, which is very similar to that obtained from molecular docking analysis (∼20 Å). The system also shows temperature-dependent reversible FRET, which may be used as a thermal sensor for the temperature-sensitive biological systems.

Preferential Binding of Epirubicin Hydrochloride with Single Nucleotide Mismatched DNA and Subsequent Sequestration by a Mixed Micelle

“…Recently, FCS has been utilized to gain insight regarding processes involving the molecular diffusion, complexation, conformational dynamics, and binding affinity of the drug to its targets such as protein, DNA, and so forth with single-molecule resolution. ,,,, It is one of the advanced techniques to estimate the diffusion properties such as the diffusion time (τ D ) and diffusion coefficient ( D t ) of the probe by measuring the fluctuation in fluorescence intensity over time. It is reported that the FCS technique has been employed to evaluate the efficiency of a drug being delivered to a target site, such as DNA. , Here, we have estimated the diffusion coefficient, hydrodynamic radius ( r H ), and binding strength of EPR-WM and EPR-MM DNA systems by monitoring the diffusion of EPR in both bound and free states at very low concentrations (10 nM).…”

“…In recent years, research has been directed toward the identification and design of site- and sequence-specific DNA-binding small-molecule drugs to make the nucleic acids a new drug discovery target. Most of the anticancer drugs bind to DNA in a more sequence-specific manner through various kinds of noncovalent interaction modes such as external binding with a phosphate backbone, groove binding, and intercalation between base pairs. − These types of noncovalent binding modes are reversible and typically preferred over covalent binding, keeping the toxic side effects and drug metabolism in mind. − Anthracycline drug epirubicin hydrochloride (EPR, Scheme a) is one of the most imperative anticancer drugs − used in the treatment of leukemia, lymphoma, and sarcoma and is reported to be less toxic compared to its parent compound doxorubicin. , In a cancer cell, it localizes inside the nucleus by forming a complex with DNA through an intercalation mode of binding. , This complex prevents topoisomerase II by interfering with protein synthesis, and hence the subsequent DNA replication stops . However, an in-depth elucidation of the sequence-selective binding interaction of EPR with DNA through a systematic study and its binding aspects toward single nucleotide mismatched base pairs containing DNA, which is overexpressed in cancer cells, is not well understood.…”

Polyethylene Glycol Crowder’s Effect on Enzyme Aggregation, Thermal Stability, and Residual Catalytic Activity

“…Industrial and biomedical applications of enzymes continue to expand because of their unique catalytic efficiency and selectivity. − Many mesophilic enzymes have poor thermal stability and poor retention of their catalytic activity above 50–60 °C, − thus limiting their applications when extended use at elevated temperatures is beneficial to increase productivity and decrease bacterial contamination of the industrial processes and when enzyme storage requires a low temperature. Simple and cost-efficient methods to improve enzyme stability are in high demand.…”

Secondary Structure-Dominated Layer-by-Layer Growth Mode of Protein Coatings

“…To study the changes in the protein secondary structure at different pH values, CD spectra were tested and analyzed. 42 The positive peak centered at 193 nm and one negative wide peak at 216 nm were observed for pH 6.0 alone (Figure 2a), suggesting that the proportion of every secondary structure is similar. For pH 7.0, a positive peak was observed at 193 nm, and two negative peaks were observed at 209 and 220 nm.…”

“…For (TA/Lyz) n , the conformation of TA was fixed, and checking the conformation of Lyz was very necessary to understand the above diffusion behavior of TA. To study the changes in the protein secondary structure at different pH values, CD spectra were tested and analyzed . The positive peak centered at 193 nm and one negative wide peak at 216 nm were observed for pH 6.0 alone (Figure a), suggesting that the proportion of every secondary structure is similar.…”