title: Energetics of solvent and ligand-induced conformational changes in alpha-lactalbumin.
abstract: The energetics of structural changes in the holo and apo forms of a-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-alpha-lactalbumin is stable below 14 C and undergoes a conformational change to a native-like molten globule intermediate at temperatures above 25 degrees C. The denaturation of either holo- or apo-alpha-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 degrees C. which is essentially invariant to protonation effects. This small enthalpy effect infers that stabilization of alpha-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 alpha-lactalbumin as a function of solvent conditions furnishes significant insight regarding the molecular flexibility and regulatory mechanism mediated by this protein.
authors: Department of Biology and Biocalorimetry Center, The Johns Hopkins University, Baltimore, Maryland 21218, USA. griko@jhunix.hcf.jhu.edu
authors: Y V
authors: YV
authors: Griko
authors: D P
authors: DP
authors: Remeta
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D012997:Solvents; D013696:Temperature; D013816:Thermodynamics
chemical_list: D008024:Ligands; D012997:Solvents; D007768:Lactalbumin; D002118:Calcium