The interaction of Bovine Serum Albumin (BSA) with limonene has been studied by UV-visible spectroscopy, fluorescence spectroscopy and molecular docking, and its effects on protein conformation, topology and stability were determined by Circular Dichroism (CD), Dynamic Light Scattering (DLS) and Differential Scanning Calorimetry (DSC). A gradual decrease in Stern–Volmer quenching constants with the increase in temperature showed the static mode of fluorescence quenching. The obtained binding constant (K_b) was ∼10⁴ M⁻¹. The temperature dependent K_b, Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) changes were calculated, which revealed that the reaction is spontaneous and exothermic. The UV-visible spectra showed a change in the peaks within the aromatic region indicating hydrophobic interactions with Trp, Tyr and Phe in the protein. Moreover, limonene induced an increase in α-helical contents probably on the cost of random coils or/and β-sheets of BSA, as observed from the far-UV CD spectra. The topology of BSA in the presence of limonene was slightly altered, as obtained from DLS results. The stability was also enhanced as revealed through thermal denaturation study by DSC and CD. Molecular docking study depicted that limonene fits into the hydrophobic pocket close to Sudlow site I in domain IIA of BSA. The present study will be helpful in understanding the binding mechanism of limonene and associated stability and conformational changes.

Different proteins have different amino acid sequences as well as conformations, and therefore different propensities to aggregate. Electrostatic interactions have an important role in the aggregation of proteins as revealed by our previous report (J. M. Khan et al., PLoS One, 2012, 7, e29694). In this study, we designed and executed experiments to gain knowledge of the role of charge variations on proteins during the events of protein aggregation with lysozyme as a model protein. To impart positive and negative charges to proteins, we incubated lysozyme at different pH values of below and above the pI (∼11). Negatively charged SDS was used to ‘antagonize’ positive charges on lysozyme. We examined the effects of pH variations on SDS-induced amyloid fibril formation by lysozyme using methods such as far-UV circular dichroism, Rayleigh scattering, turbidity measurements, dye binding assays and dynamic light scattering. We found that sub-micellar concentrations of SDS (0.1 to 0.6 mM) induced amyloid fibril formation by lysozyme in the pH range of 10.0–1.0 and maximum aggregation was observed at pH 1.0. The morphology of aggregates was fibrillar in structure, as visualized by transmission electron microscopy. Isothermal titration calorimetry studies demonstrated that fibril formation is exothermic. To the best of our current understanding of the mechanism of aggregation, this study demonstrates the crucial role of electrostatic interactions during amyloid fibril formation. The model proposed here will help in designing molecules that can prevent or reverse the amyloid fibril formation or the aggregation.

Thymoquinone (TQ) is the main constituent of Nigella sativa and is traditionally used as a folk medicine. Our aim was to investigate the binding mechanism of TQ to human serum albumin (HSA) isoforms (‘N’ form at pH 7.4 and ‘B’ form at pH 9.0) using biophysical methods such as intrinsic tryptophan fluorescence quenching, isothermal titration calorimetry (ITC), circular dichroism (CD), dynamic light scattering (DLS), Förster resonance energy transfer (FRET) and antioxidant activity in the absence and presence of TQ. We have calculated the binding and thermodynamic parameters from spectroscopic and calorimetric methods. CD and DLS were respectively used to monitor the changes in the secondary structure and hydrodynamic radii of HSA as a result of its interaction with TQ. The esterase and antioxidant or radical scavenging activities of both the isoforms of HSA were investigated in the absence/presence of TQ. The antioxidant activity of TQ was remarkably enhanced upon its interaction with HSA. Therefore, the efficiency of HSA to scavenge the free radical ions was increased in the presence of TQ which is generated in the body by various metabolic processes.

Ligand-induced conformational changes are of immense importance for the biological activity of a protein. An in-depth understanding of salutary and deleterious effects of ligand-induced conformational alterations in single- and multi-chain proteins would lend a hand in human welfare. Unlike single-chain proteins, the function of multichain proteins depends upon the inherent properties of the subunit interfaces. The interfaces of temporary oligomeric proteins and the active sites of enzymes are of similar characteristics but the interfaces are more conservative than the active sites. Therefore, these interfaces may possibly be represented as drug targets by inhibition or induction of the oligomerization process. Thus without detailed structural understanding of ligand-induced conformational changes in a protein, structure-based rational drug designing is a great challenging task. So the purpose of this review is to clarify or enlighten the reader at the degree of internal motions related to protein backbone and side-chain flexibility which occur on binding of small molecule to a protein target. This can prove helpful to improve the conformational prediction for a protein–ligand complex. Besides a detailed description of protein–ligand interaction, this review also focuses on structure–activity relationships of protein which will surely help in the rational drug designing.