Yuri V. Griko

A calorimetric study of the influence of calcium on the stability of bovine α-lactalbumin.

Abstract Bovine α-lactalbumin has been studied by differential scanning calorimetry with various concentrations of calcium to elucidate the effect of this ligand on its thermal properties. In the presence of excess calcium, α-lactalbumin unfolds upon heating with a single heat-absorption peak and a significant increase of heat capacity. Analysis of the observed heat effect shows that this temperature-induced process closely approximates a two-state transition. The transition temperature increases in proportion with the logarithm of the calcium concentration, which results in an increase in the transition enthalpy as expected from the observed heat capacity increment of denaturation. As the total concentration of free calcium in solution is decreased below that of the proteins, there are two temperature-induced heat absorption peaks whose relative area depends on the calcium concentration, such that further decrease of calcium concentration results in a increase of the low-temperature peak and a decrease of the high-temperature one. The high-temperature peak occurs at the same temperature as the unfolding of the holo-protein, while the low-temperature peak is within the temperature range associated with the unfolding of the apo-protein. Statistical thermodynamic modeling of this process shows that the bimodal character of the thermal denaturation of bovine α-lactalbumin at non-saturated calcium concentrations is due to a high affinity of Ca 2+ for α-lactalbumin and a low rate of calcium exchange between the holo- and apo-forms of this protein. Using calorimetric data, the calcium-binding constant for α-lactalbumin has been determined to be 2.9×10 8 M −1 .

Energetics of Ca2+–EDTA interactions: calorimetric study

The interaction between Ca(2+) and EDTA has been studied using isothermal titration calorimetry to elucidate the detailed mechanism of complex formation and to relate the apparent thermodynamic parameters of calcium binding to the intrinsic effects of ionization. It has been shown that Ca(2+) binding to EDTA is an exothermic process in the temperature range 5-48 degrees C and is highly dependent on the buffer in which the reaction occurs. Calorimetric measurements along with pH-titration of EDTA under different solvent conditions shows that the apparent enthalpy effect of the binding is predominantly from the protonation of buffer. Subtraction of the ionization effect of buffer from the total enthalpy shows that the enthalpy of binding Ca(2+) to EDTA is -0.56 kcal mol(-1) at pH 7.5. The DeltaH value strongly depends on solvent conditions as a result of the degree of ionization of the two amino groups in the EDTA molecule, but depends little on temperature, indicating that the heat capacity increment for metal binding is close to zero. At physiological pH values where the amino groups of EDTA with pK(a)=6.16 and pK(a)=10.26 are differently ionized, the coordination of the Ca(2+) ion into the complex leads to release of one proton due to deprotonation of the amino group having pK(a)=10.26. Increasing the pH up to 11.2, where little or no ionization occurs, leads to elimination of the enthalpy component due to ionization, while its decrease to pH 2 leads to its increase, due to protonation of the two amino groups. The heat effect of Ca(2+)/EDTA interactions, excluding the deprotonation enthalpy of the amino groups, i.e. that associated with the intrinsic enthalpy of binding, is higher in value (Delta(b)H(o)=-5.4 kcal mol(-1)) than the apparent enthalpy of binding. Thus, the large DeltaG value for Ca(2+) binding to EDTA arises not only from favorable entropic but also enthalpic changes, depending on the ionization state of the amino groups involved in coordination of the calcium. This explains the great variability in DeltaH obtained in previous studies. The ionization enthalpy is always unfavorable, and therefore dramatically decreases Ca(2+) affinity by reduction of the enthalpy term of the stability function. The origin of the enthalpy and entropy terms in the stability of the Ca(2+)-EDTA complex is discussed.

Thermal and urea-induced unfolding in T7 RNA polymerase: calorimetry, circular dichroism and fluorescence study.

Structural changes in T7 RNA polymerase (T7RNAP) induced by temperature and urea have been studied over a wide range of conditions to obtain information about the structural organization and the stability of the enzyme. T7RNAP is a large monomeric enzyme (99 kD). Calorimetric studies of the thermal transitions in T7RNAP show that the enzyme consists of three cooperative units that may be regarded as structural domains. Interactions between these structural domains and their stability strongly depend on solvent conditions. The unfolding of T7RNAP under different solvent conditions induces a highly stable intermediate state that lacks specific tertiary interactions, contains a significant amount of residual secondary structure, and undergoes further cooperative unfolding at high urea concentrations. Circular dichroism (CD) studies show that thermal unfolding leads to an intermediate state that has increased β‐sheet and reduced α‐helix content relative to the native state. Urea‐induced unfolding at 25°C reveals a two‐step process. The first transition centered near 3 M urea leads to a plateau from 3.5 to 5.0 M urea, followed by a second transition centered near 6.5 M urea. The CD spectrum of the enzyme in the plateau region, which is similar to that of the enzyme thermally unfolded in the absence of urea, shows little temperature dependence from 15° to 60°C. The second transition leads to a mixture of poly(Pro)II and unordered conformations. As the temperature increases, the ellipticity at 222 nm becomes more negative because of conversion of poly(Pro)II to the unordered conformation. Near‐ultraviolet CD spectra at 25°C at varying concentrations of urea are consistent with this picture. Both thermal and urea denaturation are irreversible, presumably because of processes that follow unfolding.

Denaturation versus unfolding: energetic aspects of residual structure in denatured alpha-lactalbumin.

Denaturational changes in α-lactalbumin result in different degrees of disordering of the protein molecule. The thermally denatured states have been studied to elucidate the energetics of residual structure and its contributions to the stability of the native conformation. The value of the heat capacity increment of α-lactalbumin denaturation correlates closely with the amount of residual secondary structure in the denatured protein, therefore reflecting the degree of its disordering and accessibility to solvent. As a result of the observed correlation, the behavior of protein denaturation functions is influenced by the degree of disordering of protein conformation in the denatured state. Analysis of the calorimetric data shows that the denaturational transition of α-lactalbumin is described by different thermodynamic functions when it proceeds to an ordered compact denatured state and to the disordered unfolded state. This difference is related to unfolding of the compact denatured state known as a molten globule state, which is populated differently under different denaturing conditions. The enthalpy and entropy of the transition from the native to the compact denatured state are always higher in magnitude than the enthalpy and entropy of the complete unfolding reaction due to the large negative hydration effect upon molten globule unfolding. Since the hydration effect increases with decreasing temperature, the gap between the partial denaturing and complete unfolding thermodynamic parameters also increases, resulting in a large difference at physiological temperatures. The results clearly indicate that a degree of residual structure in the denatured state must be taken into account to yield a more accurate description of protein structural energetics.