Microscopic Investigation Of Conformational Stability Of Proteins In Amino Acid Solutions And The Properties Of Solvent Around It

Abstract

To perform biological functions and avoid unwanted immunological reactions, protein’s threedimensional folded native forms needed to be preserved. Under harsh environmental conditions, a protein may undergo loosening of secondary segments and attend a denatured state. The use of additives in the aqueous solution of protein is a common practice to preserve the native-like structure. The prime objective of this thesis has been to explore the effects of different amino acids as additives on regulating the conformational and solvation properties of protein at ambient and in thermally stressed conditions. To fulfill the objectives, extensive atomistic molecular dynamics (MD) simulations with two proteins, insulin monomer, and ubiquitin, were carried out with amino acid solutions. The thesis consists of six chapters. In Chapter 1, a brief discussion has been enlisted on the current status of knowledge and recent development in this area, along with the methodologies adopted in this thesis. In Chapter 2, the comparative efficiency of three basic amino acids, arginine, histidine, and lysine, on regulating the conformational flexibilities of insulin monomer at ambient conditions was investigated. The study revealed that the relative more loss of configurational entropy of insulin in arginine solution than in the pure water and other two amino acid solutions was due to the presence of motionally bound, less entropic hydration water around insulin in arginine solution than in histidine/lysine solution. This instigated choosing arginine as one of the most promising preservative additives (osmolyte) among the three at ambient temperature. As a result, in Chapter 3 influence of arginine concentrations in modulating insulin conformations at ambient and elevated temperatures was studied thoroughly by conventional MD and replica-exchange molecular dynamics (REMD) approaches. To identify the physical origin of the arginine concentration dependent differential stability of insulin in solution, a detailed investigation on insulin-water and insulin-arginine interactions was carried out in Chapter 4. The study showed that the exclusion of arginine from the protein surface increases the local structuration of water around it. The favourable formation of a sluggish water-arginine mixed solvation layer at a higher arginine concentration of 2 M helps to maintain the structural rigidity of the protein. Further, it has been observed that arginine stabilizes the protein through several aromatic interactions; cation-pi/anion-pi, as well as hydrogen-bonded interactions. Motivated by this, the relative effects of aromatic amino acids, phenylalanine, tyrosine, and tryptophan solutions, on insulin’s conformational properties were studied in Chapter 5. The calculations revealed that while tryptophan was prone to interact with the protein through cation-pi interactions, phenylalanine and tyrosine preferred pi-pi stacking. In Chapter 6, the preserving efficacy of the 2 M arginine solution has been tested by considering another model protein, ubiquitin. Such a study would facilitate establishing the general idea about the preservation efficiency of this solution. In this work, the conformational stability of the secondary structural segments of ubiquitin at ambient and elevated temperatures was studied through combined MD and REMD techniques. This chapter unfolds the detailed story of the structural transitions of ubiquitin and the reason behind the restricted hydrogen-bond dynamics of protein-water and protein-arginine solutions that primarily helped to conserve protein’s native folded form firmly.