Stability and physicochemical properties of the bovine brain phosphatidylethanolamine-binding protein

The equilibrium behaviour of the bovine phosphatidylethanolamine-binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far-UV and near-UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of beta-sheets, with Trp residues mostly localized in a hydrophobic environment; these results suggest that the conformation of PEBP in solution is similar to the three-dimensional structure determined by X-ray crystallography. The pH-induced conformational changes show a transition midpoint at pH 3.0, implying nine protons in the transition. At neutral pH, the thermal denaturation is irreversible due to protein precipitation, whereas at acidic pH values the protein exhibits a reversible denaturation. The thermal. denaturation curves, as monitored by CD, fluorescence and differential scanning calorimetry, support a two-state model for the equilibrium and display coincident values with a melting temperature T-m = 54 degrees C, an enthalpy change Delta H = 119 kcal.mol(-1) and a free energy change Delta G(H2O, 25 degrees C) = 5 kcal.mol(-1). The urea-induced unfolding profiles of PEEP show a midpoint of the two-state unfolding transition at 4.8 M denaturant, and the stability of PEBP is 4.5 kcal.mol(-1) at 25 degrees C. Moreover, the surface active properties indicate that PEEP is essentially a hydrophilic protein which progressively unfolds at the air/water interface over the course of time. Together, these results suggest that PEEP is well-structured in solution but that its conformation is weakly stable and sensitive to hydrophobic conditions: the PEEP structure seems to be flexible and adaptable to its environment.