Energetics of solvent and ligand-induced conformational changes in α-lactalbumin

Abstract

The energetics of structural changes in the holo and apo forms of α-lactalbumin and the transition between their native and denatured states induced by binding Ca²⁺ and Na⁺ have been studied by differential scanning and isothermal titration microcalorimetry and circular dichroism spectroscopy under various solvent conditions. Removal of Ca²⁺ from the protein enhances its sensitivity to pH and ionic conditions due to noncompensated negative charge–charge interactions at the cation binding site, which significantly reduces its overall stability. At neutral pH and low ionic strength, the native structure of apo-α-lactalbumin is stable below 14 °C and undergoes a conformational change to a native-like molten globule intermediate at temperatures above 25 °C. The denaturation of either holo- or apo-α-lactalbumin is a highly cooperative process that is characterized by an enthalpy of similar magnitude when calculated at the same temperature. Measured by direct calorimetric titration, the enthalpy of Ca²⁺-binding to apo-LA at pH 7.5 is −7.1 kJ mol⁻¹ at 5.0 °C, which is essentially invariant to protonation effects. This small enthalpy effect infers that stabilization of α-lactalbumin by Ca²⁺ is primarily an entropy driven process, presumably arising from electrostatic interactions and the hydration effect. In contrast to the binding of calcium, the interaction of sodium with apo-LA does not produce a noticeable heat effect and is characterized by its ionic nature rather than specific binding to the metal-binding site. Characterization of the conformational stability and ligand binding energetics of α-lactalbumin as a function of solvent conditions furnishes significant insight regarding the molecular flexibility and regulatory mechanism mediated by this protein.