Protein stability and conformation: Impact of small molecules and cytoplasmic constituents

Abstract

A variety of small molecules are either directly or indirectly consumed by our body through various sources. These small molecules might cause changes in the biological processes upon their interaction with different proteins present inside the body. Therefore, investigation of the interaction between various types of small molecules and proteins is crucial owing to its biomedical implications to understand the binding pattern and changes in structure and functions of proteins under physiological conditions. Additionally, the interaction of small molecules also shows the ability to regulate the formation of amyloid fibrils. Beside the small molecules, cellular components and parameters like temperature, salt ions, chaperones, crowding agents, pH, solvent etc. also could control protein folding mechanism. Hence, this thesis aims to study the effects of different small molecules and the cytoplasmic constituents on the stability and conformation of different proteins with biological relevance. The overall thesis work is focused on three objectives. The first objective of the thesis was further divided into two parts. The first part described the interaction of synthetic dye molecules with globular protein (Lysozyme, ‘Lyz’) and its effect on protein conformation and stability. Conformational changes in Lysozyme were induced upon its interaction with the selected dye molecules. In the second part, the impact of an anticancer drug (5-fluorouracil, 5-Fu) on conformation, stability, and activity of Lysozyme was investigated using different spectroscopic and computational methods. Our results revealed that the binding between drug and Lysozyme caused moderate alterations in the protein conformation and the stability. In the second objective, both the interaction and in vitro fibrillation study of Lysozyme in the presence of food colorants (Sunset Yellow (SY) and Ponceau 4R (P4R)) were studied. The binding results suggested that P4R has a remarkably higher affinity for Lyz in comparison to SY. However, results from the in vitro fibrillation illustrated that SY and P4R inhibited the formation of amyloid fibril. In the third objective, the conformation of the model protein, CRABP I (cellular retinoic acid binding protein I), was investigated as a function of micro-environment in the presence of several anions, such as SO4, HPO4, and Cl using fluorescence-based techniques. FCS observations revealed the impact of anions on the diffusion of protein as well as the hydrodynamic radii at room temperature. Additionally, fluorescence (steady-state and lifetime) and circular dichroism (CD) results indicated the conformational changes in the protein. Thus, the findings from the above studies suggested that the addition of small molecules and salt ions significantly influence the conformation and stability of proteins. We hope that the information obtained from this thesis will be helpful to develop and design new compounds that can efficiently regulate the specific cellular pathways and biological processes.