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

The equilibrium behaviour of the bovine phosphatidylethanolamine-binding pr otein (PEBP) has been studied under various conditions of pH, temperature a nd urea concentration. Far-UV and near-UV CD, fluorescence and Fourier tran sform infrared spectroscopies indicate that, in its native state, PEBP is m ainly composed of beta-sheets, with Trp residues mostly localized in a hydr ophobic 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 m idpoint at pH 3.0, implying nine protons in the transition. At neutral pH, the thermal denaturation is irreversible due to protein precipitation, wher eas at acidic pH values the protein exhibits a reversible denaturation. The thermal. denaturation curves, as monitored by CD, fluorescence and differe ntial scanning calorimetry, support a two-state model for the equilibrium a nd 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 Del ta G(H2O, 25 degrees C) = 5 kcal.mol(-1). The urea-induced unfolding profil es of PEEP show a midpoint of the two-state unfolding transition at 4.8 M d enaturant, and the stability of PEBP is 4.5 kcal.mol(-1) at 25 degrees C. M oreover, 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 sensi tive to hydrophobic conditions: the PEEP structure seems to be flexible and adaptable to its environment.