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 CO−dissociation from NCO (NCO→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 CO−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. log kdiss is found to be decreased with increasing solvent composition (glycerol, sucrose, and glucose) and viscosity, indicating that the solvent viscosity controls the internal dynamics of NCO. At a given particular concentration, dextran 70 has always higher viscosity than dextran 40 so the greater decrease of log kdiss for dextran 70 than that of the dextran 40 suggests that the viscosity of crowding agent also plays a vital role in controlling the internal dynamics of NCO and MbCO. The thermal or denaturant unfolding transitions measured for Ferrocyt c by different optical probes (CD, fluorescence and absorbance) and common optical probes with multiple wavelengths are within error nearly super imposable and cooperative. The observation of indistinct thermal or denaturant unfolding transition curves for Ferrocyt c do not reveal the accumulation of any equilibrium structural intermediate to a detectable level, which suggests that the thermal and denaturant-induced unfolding of Ferrocyt c occur in two-state manner. Two-state thermodynamic analysis of chemical (GdnHCl or urea) and thermal unfolding transitions of native Ferrocyt c (N-state) and Mb carried out in the absence and presence of 200 mg/ml dextran 40, dextran 70 and ficoll 70 reveals that the smaller size crowder (dextran 40) has a greater impact on the thermodynamic stability of native Ferrocyt c. The free amino acids and their derivatives are the naturally occurring osmolytes. The free amino acids in cytoplasm can influence the functional properties of protein. Chapter 5 investigated the role of amino acids on stability, folding, and internal dynamics of native cytochrome c (Cyt c) and myoglobin (Mb). To determine the role of amino acids on the internal dynamics of native-like compact state (NCO) and carbonmonoxymyoglobin (MbCO), the kinetic and thermodynamic parameters for CO-dissociation from NCO (NCO→N+CO) and CO-replacement from MbCO by hexacyanoferrate ion were measured at varying concentrations of L-amino acids (alanine, arginine, glycine, proline, serine, and threonine) at pH 7.0. As [amino acids] is increased, the CO−dissociation reaction of NCO and CO−replacement reaction of MbCO are decelerated, indicating that the amino acids presence in the reaction medium reduce the structural fluctuations responsible for CO dissociation from NCO and CO replacement from MbCO. The amino acid−mediated reduction in structural fluctuations of NCO and MbCO typically follow the order: arginine> serine> proline> glycine> alanine >threonine. Two-state thermodynamic analysis of chemical (GdnHCl or urea) and thermal unfolding transitions of native Ferrocyt c (N-state) and Mb carried out in the presence of various concentrations of amino acids (alanine, arginine, glycine, proline, serine, and threonine) reveals that alanine, glycine, proline, serine, and threonine presence in reaction medium increase the thermodynamic stability of Ferrocyt c and Mb but in the presence of arginine decreases the thermodynamic stability of these proteins. The amino acid-mediated increase in thermodynamic stability for Ferrocyt c and Mb typically follow the order: serine> glycine> alanine >threonine>proline. The decrease in thermodynamic stability of Ferrocyt c and Mb in the presence of arginine is presumably because of the presence of guanidium group (Gdn+) in the arginine. While the effects of denaturants (urea and GdnHCl) on the internal dynamics of native Cyt c and Mb have been extensively investigated, but the effect of crowding agents and amino acids on the internal dynamics of natively folded carbonmonoxycytochrome c (NCO) in the presence of varying concentration denaturants, across the folding-unfolding transition are not explored so far. Chapter 4 and Chapter 6 investigated the effect of crowding agents and amino acids, respectively, on the denaturant-dependent internal dynamics of NCO and thermodynamic stability of native Cyt c and Mb at pH 7.0. Analysis of kinetic and thermodynamic parameters measured for CO-dissociation reaction of NCO at different concentrations of GdnHCl or urea in the absence and presence of fixed concentrations of crowding agents (dextran 40, dextran 70 and ficoll 70) and L-amino acids (alanine, arginine, glycine, proline, serine, and threonine) at pH 7.0 provides three important information: (i) in subdenaturing region, crowding agents or L-amino acids presence in reaction medium exhibits an additive effect on the denaturant-mediated reduction in structural fluctuations responsible for CO dissociation, which typically follow the order for amino acids as : (arginine> serine> proline> glycine> alanine >threonine) and crowding agents as: (dextran 70> dextran 40> Ficoll 70), (ii) in denaturing region, the crowding agents or amino acids presence in reaction medium counteract the structural fluctuations responsible for unfolding the protein, and (iii) the structural fluctuation that unfolds the protein are found to be more opposed by the larger sized and anisotropic geometry shaped crowding agents (dextran 70> dextran 40> Ficoll 70) and larger sized and lesser hydrophobic amino acids (arginine> serine> proline> glycine> alanine >threonine). Two-state thermodynamic analysis of thermal and urea unfolding curves of Cyt c and Mb measured at different concentrations of GdnHCl in the absence and presence of fixed concentrations of crowding agents (dextran 40, dextran 70 and ficoll 70) and L-amino acids (alanine, arginine, glycine, proline, serine, and threonine) at pH 7.0 provides three important information: (i) crowding agents presence in reaction medium counteracts the deleterious effect of denaturants on stability of proteins and they typically follow the order (dextran 40> dextran 70> ficoll 70), (ii) alanine, glycine, proline, serine and threonine counteract the deleterious effect of denaturants on stability of proteins and they typically follow the order: serine> glycine>alanine>threonine>proline, and (iii) at lower concentrations of GdnHCl, L-arginine show an additive effect on the deleterious effect of denaturant on stability of proteins while at higher concentrations of GdnHCl, it counteract the deleterious effect of denaturants. The folding of protein, an important process for protein to fulfill normal functions, takes place in crowded physiological environments. Chapter 7 characterized the structural, kinetic and thermodynamic properties of the macromolecular crowding-induced molten globule states of the alkali pH-denatured proteins. Near-UV CD, far-UV CD, tryptophan fluorescence and 1-anilino-8-napthalene sulfonate (ANS) binding experiments of base-denatured Cyt c, apoMb and Lyz carried out in the absence and presence of different concentrations of crowding agents (dextran 70 and ficoll 70) indicate that the crowders-induced fully populated conformations are molecular compact states containing native-like secondary structural contents but disordered tertiary interactions. Thermodynamic analysis of the far UV-CD (222 nm) monitored thermal denaturation curves of base-denatured Cyt c, apoMb and Lyz measured in the absence and presence of 300 mg ml-1 of crowding agents (dextran 70 and ficoll 70) suggests that the crowding agent presence in the reaction medium increase the thermal stability of the base-denatured proteins. Kinetic and thermodynamic experiments involving the measurement of the CO-association to the base−denatured Ferrocyt c (pH 12.9 (±0.1)) in the absence and presence of different concentrations of crowding agents (dextran 70 and ficoll 70) indicate that the presence crowding agents in the reaction medium constrains the internal dynamics of base−denatured Ferrocyt c. While the effects of salt and alcohol on the structural and thermodynamic properties of native and acid pH-denatured proteins are extensively studied, the effects of these additives on structural, kinetics and thermodynamic properties of base-denatured proteins are not explored so far. Chapter 8 characterized the structural, kinetic and thermodynamic properties of the cations (NaCl, KCl and CsCl) and TFE (2,2,2−trifluoroethanol)-induced molten globule states of the alkali pH-denatured proteins. Near-UV CD, far-UV CD, tryptophan fluorescence and 1-anilino-8-napthalene sulfonate (ANS) binding experiments of base-denatured Cyt c, apoMb and Lyz carried out in the absence and presence of different concentrations of NaCl, KCl, CsCl and TFE revealed that the cations (chloride salt of Na+, K+ and Cs+) and TFE-induced fully populated conformations are molecular compact states containing native-like secondary structural contents but disordered tertiary interactions. Thermodynamic analysis of the far UV-CD (222 nm) monitored thermal denaturation curves of base-denatured Cyt c, apoMb and Lyz measured in the absence and presence of 1.0 M NaCl, KCl and CsCl suggests that the cations (chloride salt of Na+, K+ and Cs+) presence in the reaction medium increase the thermal stability of the base-denatured proteins. Kinetic and thermodynamic experiments involving the measurement of the CO-association to the base−denatured Ferrocyt c (pH 12.9 (±0.1)) in the absence and presence of different concentrations of TFE and salt (NaCl, KCl and CsCl) indicate that the TFE and cations (Na+, K+ and Cs+) presence in the reaction medium constrain the internal dynamics of base−denatured Ferrocyt c. Transferrins (Tfs; serum transferrin (sTf), ovotransferrin (oTf), and lactoferrin (Lf)) play the major roles in the iron metabolism of vertebrates, and some invertebrates. The iron coordination is distorted octahedral with four ligands provided by amino acid residues (two residue of tyrosine, one aspartic acid, and one histidine) and the remaining coordinates is shared by a synergistic anion. Carbonate is the main synergistic anion in-vivo but when carbonate is absent, other small organic molecules such as oxalate, malonate, glycine etc can function as synergistic anion. Chapter 9 investigated the effects of substitution of oxalate anion for carbonate anion on the stability and iron release dynamics of oTf and sTf. Analysis of the pH-, urea- and thermal-denaturations profiles of iron release (based on absorbance (465 nm)) for oxalate anion bound diferric-sTf/oTf (Fe2sTf/Fe2oTf) reveals that the carbonate bound Fe2Tfs binds Fe3+ less tightly than the oxalate bound Fe2Tfs. Thermodynamic analysis of the near- UV CD (282nm) or far-UV CD (222 nm) monitored thermal and urea-induced unfolding curves for these two different synergistic anions bound Fe2sTf and Fe2oTf reveals that the carbonate bound Fe2sTfs has relatively lesser structural stability than oxalate bound Fe2sTfs. Differential scanning calorimetric study of these two synergistic anions bound Fe2sTf reveals that the carbonate bound Fe2sTfs has relatively less thermal stability than oxalate bound Fe2sTfs. Kinetic and thermodynamic parameters involving measurement of the reductive iron release (Fe2+ release) and urea-denaturation induced iron release (Fe3+ release) reactions for carbonate and oxalate bound monoferric N-lobe of sTf and oTf (FeNsTf and FeNoTf) at pH 7.4 and 5.6 reveal that the substitution of carbonate by oxalate retards the iron release from FeNTfs with a increase in enthalpic barrier.