Kinetic and Thermodynamic Studies on the Effect of Amino Acids Alcohols Crowding Agents and Lyotropic Salts on Proteins

Abstract

About 10 to 40% of cytoplasm volume is generally occupied by macromolecules viz. proteins, carbohydrates, nucleic acids etc. The highly crowded conditions found in cytoplasm can affect the thermodynamic and kinetic properties of proteins. Chapter 3 investigated the role of macromolecular crowding on stability, folding, and internal dynamics of native cytochrome c (Cyt c) and myoglobin (Mb). Carbonmonoxycytochrome c (Cyt-CO) refolds to a native-like compact state (NCO-state), where the non-native Fe2+-CO interaction persists. Slow thermal-dissociation of CO transforms the NCO-state to native-state (N-state), where the native Fe2+-M80 bond recovers. To determine the role of macromolecular crowding on the internal dynamics of NCO and carbonmonoxymyoglobin (MbCO), the kinetic and thermodynamic parameters for COand#8722;dissociation from NCO (NCOand#8594;N+CO) and CO-replacement from MbCO by hexacyanoferrate ion were measured at varying concentrations of crowding agents (dextran 70, dextran 40, ficoll 70) and viscogens (glycerol, sucrose, and glucose). As [crowding agent] is increased, the rate coefficients of COand#8722;dissociation for NCO (kdiss) and CO-replacement for MbCO (koff) decrease exponentially. The values of log kdiss and log koff are found to be decreased more for dextran 70 than that of the ficoll 70, suggesting that the shape of crowding agent plays an important role in controlling the internal dynamics of NCO and MbCO. log kdiss and log koff are also found to be decreased more for dextran 70 than that of dextran 40. Dextran 70 has the larger size than that of the dextran 40, so the greater decrease of log kdiss and log koff for dextran 70 suggests that the size of crowding agent also plays a significant role in controlling the internal dynamics of NCO and MbCO. A general approach for investigating the importance of protein dynamics in a chemical reaction is to determine the role of solvent viscosity on reaction rate.