Modulating Protein Stability and Dynamics by Osmolytes and Electrolytes

Abstract

In chapter 3, the effects of urea and alkylureas were investigated on thermodynamic stability and internal dynamics of heme proteins (Cyt c and Mb). To determine the effects of urea and alkylureas on the internal dynamics of heme proteins, the kinetic and thermodynamic parameters for CO-association reaction of Ferrocyt c and CO-replacement reaction of MbCO by hexacyanoferrate ion were measured under varying concentrations of urea and alkylureas (MU, DMU, EU, TMU) at pH 7.0. As [denaturant] is increased, the rate coefficient of CO-association for Ferrocyt c ( k ass ) first decrease in subdenaturing region and then increase on going from subdenaturing to denaturing milieu, which indicates that the low concentrations of denaturants constrain the internal dynamics of Ferrocyt c . Within the subdenaturing limit, the denaturant-mediated constrained dynamics of Ferrocyt c is found to be more for urea and least for TMU. However, within the subdenaturing limit, such denaturant-mediated constrained dynamics is not observed for Mb. Intermolecular docking between horse Cyt c and denaturant molecule (urea, MU, DMU, EU and TMU) reveals that polyfunctional interactions between the denaturant and different groups of Ω-loop of Cyt c and other part of protein decrease with an increase of alkyl group on urea molecule, which suggests that the decrease in the extent of restricted dynamics of Ω-loop with a corresponding increase of alkyl groups on urea molecule is due to the decrease of denaturant-mediated cross-linking interactions. These denaturant mediated interactions are expected to reduce the entropy of Ferrocyt c . Analysis of rate temperature data shows a progressive decrease in entropy of Ferrocyt c in the native to subdenaturing region. Thermodynamic analysis of denaturant (urea, MU, DMU, EU and TMU) effects on the thermal unfolding of Ferrocyt c and Mb reveals that (i) thermodynamic stability of proteins decreases with increasing concentration of denaturant or hydrophobicity of urea derivatives, (ii) water activity plays an important role in stabilization of protein, and (iii) destabilization of Ferrocyt c and Mb by denaturant occurs through the disturbance of hydrophobic interactions and hydrogen-bonding. In chapter 4, the effects of glycerol and trehalose were investigated on the structural, kinetic and thermodynamic properties of alkali pH-denatured Cyt c (U B -state). Near-UV CD, far-UV CD, tryptophan fluorescence and 1-anilino-8-napthalene sulfonate (ANS) binding experiments suggest that the glycerol and trehalose transform the base-denatured Cyt c to MG-states. The glycerol and trehalose -induced fully populated G B (glycerol-induced) and T B (trehalose-induced) conformations of base-denatured Ferricyt c and Cyt-CO are molecular compact states containing native-like secondary structural contents but disordered tertiary interactions. The calculated values of free energy change (  G o ) for the U B →G B (  G o ~1.76 Kcal mol -1 ) and U B →T B (  G o ~1.62 Kcal mol -1 ) transitions are within error almost same, where G B and T B are the glycerol and trehalose induced MG states of base-denatured Ferricyt c . Both G B and T B -states of Ferricyt c undergo highly cooperative thermal unfolding transitions and they show cold denaturations at low sugar concentration. As [sugar] is increased, the thermal denaturation temperature increase and the cold denaturation temperature decrease. Thermal unfolding of G B and T B states of Ferricyt c are characterized by a large heat capacity change, indicating that the hydrophobic effect also contributes substantially toward the energetic stabilization of G B and T B states. Kinetic and thermodynamic parameters associated with the measurement of CO-association reactions of alkaline Ferrocyt c (pH 12.9) at variable concentrations of glycerol and trehalose indicate substantially restricted overall motion and stiffness of the polypeptide chain in G B - and T B -states of Cyt-CO. In chapter 5, the effect of sugars (glycerol, ribose, glucose, maltose, sucrose and trehalose) was investigated on the structural and thermodynamic properties of Lyz at pH 2.3 and pH 13. Near- UV CD, far-UV CD and ANS binding experiments suggest that the glycerol and trehalose transform the base-denatured Lyz (pH 13) to MG-state. This chapter also evaluated the effect of sugars (glycerol, ribose, glucose, maltose, sucrose and trehalose) on the thermodynamic stability of Lyz at pH 2.3 both in the absence and presence of denaturants (GdnHCl and urea). Thermodynamic analysis of thermal and denaturant-induced unfolding transitions of Lyz at pH 2.3 measured at different concentrations of sugars (glycerol, ribose, glucose, maltose, sucrose and trehalose) reveals that these sugars increase the thermal and conformational stability of Lyz. Among the sugars used, the thermal and conformational stability of Lyz is increased more for trehalose and least for glycerol (trehalose > sucrose > maltose > glucose > ribose > glycerol). Thermodynamic analysis of thermal and urea-induced unfolding transitions of Lyz at pH 2.3 measured at different concentration of GdnHCl or urea in the absence and presence of fixed concentration of sugars (glycerol, ribose, glucose, maltose, sucrose and trehalose) reveals that these sugars counteract the destabilizing effect of the denaturants. The counteraction effect of sugars on the destabilizing effect of the denaturants is more pronounced for trehalose and least for glycerol (trehalose > sucrose > maltose > glucose > ribose > glycerol). In chapter 6, the effects of pH and electrolytes were investigated on the stability and iron release kinetics of oTf. The role of electrostatic interactions to the stability of iron binding to oTf have been assessed by equilibrium experiments that measure iron retention level of diferric- ovotransferrin (Fe 2 oTf) as a function of pH and urea in the presence of varying types and concentrations of salts (NaCl, Na 2 SO 4 , NaBr and NaNO 3 ) at 25  C. As [salt] is increased, the pH- midpoint for iron release increases monoexponentially and plateau at ~0.4(±0.05)M NaCl/NaBr/NaNO 3 or ~0.15(±0.03)M Na 2 SO 4 . However, at pH 7.4, the urea-midpoints for iron release (based on fluorescence emission at 340 nm) and for unfolding of Fe 2 oTf and apo- ovotransferrin (based on ellipticity values at 222 and 282 nm) are found to decrease at low salts concentrations (  0.1(±0.02)M Na 2 SO 4 or ≤0.35(±0.15) M NaCl) but are increased at higher salts concentrations. Furthermore, Na 2 SO 4 has a greater effect than does NaCl in increasing the urea- midpoints for iron release and unfolding. These results indicate that at low salt concentrations, the electrostatic interactions control the stability of the oTf-Fe 3+ complex and secondary and tertiary structures of protein, while at higher salt concentrations; salt ions behave according to Hofmeister series. At pH 5.6, as [salt] is increased, the rate constants for reductive iron release (Fe 2+ release) and urea-denaturation induced iron release (Fe 3+ release) from the N-lobe of oTf (Fe N oTf) increase monoexponentially and plateau at ~0.4(±0.1)M NaNO 3 /NaCl or ~0.2(±0.05) M Na 2 SO 4 . These results suggest that the conformational change-induced by anion binding as well as the electrostatic screening of surface Coulombic interactions plays important role in the accelerating of Fe 2+ and Fe 3+ release from Fe N oTf at endosomal pH conditions. In chapter 7, the effects of concentration, size, shape, and viscosity of crowding agents on stability and iron release kinetics of sTf-Fe 3+ complex at physiological pH 7.4 and endosomal pH 5.5-5.7 were investigated. As [crowding agent] is increased, the pH-midpoints for iron release of Fe 2 sTf shift towards the lower pH values while the urea-denaturation midpoints for iron release and unfolding of Fe 2 sTf shift towards the higher urea concentrations, which suggest that the crowding agent presence in the reaction medium increase the Tf-Fe 3+ complex and structural stability of Fe 2 sTf. Furthermore, the crowding agent mediated increase in Tf-Fe 3+ complex and structural stability of Fe 2 sTf typically follows the order: dextran 70 (rod shaped) > dextran 40 (rod shaped) > ficoll 70 (spherical shaped), which suggests that size, shape and viscosity of crowding agent also control the Tf-Fe 3+ complex and structural stability of Fe 2 sTf. As [NaCl] is increased both in the absence and presence of dextran 40, the pH-midpoints for iron release from Fe 2 sTf shift towards the higher pH values while the urea-denaturation midpoints for iron release and unfolding of Fe 2 sTf at pH 7.4 and 5.7 shift towards the lower urea concentrations, which suggest that the salt presence both in the absence and presence of crowding agent decrease the Tf-Fe 3+ complex and structural stability of Fe 2 sTf. At pH 7.4 and pH 5.5, as [crowding agent] is increased, the rate constants for reductive iron release (Fe 2+ release) and urea denaturation-induced iron release (Fe 3+ release) from the N-lobe of sTf (Fe N sTf) decrease, which suggest that the crowding agent presence in the reaction medium retards the iron release from Fe N sTf. Furthermore, the crowding agent mediated retardation in iron release typically follows the order: dextran 70 > dextran 40 > ficoll 70, which suggest that the size, shape and viscosity of crowding agents control the iron release from Fe N sTf. The anion mediated acceleration of reduction and urea denaturation induced iron release from Fe N sTf is also observed both in the absence and presence of crowding agent. In chapter 8, the mechanism of iron release from diferric ovotransferrin (Fe 2 oTf) was evaluated at mildly acidic pH (3.9  pH  4.3) in the presence of nonsynergistic anions (Cl  , SO 4 2  ). In vitro, iron release from Fe 2 oTf at mildly acidic pH in the presence of nonsynergistic anions occurs in at least six kinetically detectable steps. Step 1 involves the proton-assisted loss of the synergistic carbonate anion. In subsequent steps, iron release is controlled by slow proton transfers and anion binding. In Step 2, the N-lobe gains one proton. In Step 3, the N-lobe gains one proton with kinetic linkage to the binding of two monoanions or one dianion. In step 4, iron is released from the N-lobe with kinetic linkage to the uptake of two protons accompanied by the loss of anions. In Step 5, the C-lobe gains one proton with kinetic linkage to the binding of two mono anions or one dianion. In Step 6, iron release from the C-lobe occurs with the gains of two protons accompanied by the loss of anions.