Conformational Stability of the NH(2)-Terminal Propeptide of the Precursor of Pulmonary Surfactant Protein SP-B

Assembly of pulmonary surfactant lipid-protein complexes depends on conformational changes coupled with proteolytic maturation of proSP-B, the precursor of pulmonary surfactant protein B (SP-B), along the surfactant biogenesis pathway in pneumocytes. Conformational destabilization of the N-terminal propeptide of proSP-B (SP-B(N)) triggers exposure of the mature SP-B domain for insertion into surfactant lipids. We have studied the conformational stability during GdmCl- or urea-promoted unfolding of SP-B(N) with trp fluorescence and circular dichroism spectroscopies. Binding of the intermediate states to bis-ANS suggests their molten globule-like character. ΔG(0)(H2O) was ~ 12.7 kJ·mol(-1) either with urea or GdmCl. None of the thermal transitions of SP-B(N) detected by CD correspond to protein unfolding. Differential scanning calorimetry of SP-B(N) evidenced two endothermic peaks involved in oligomer dissociation as confirmed with 2 M urea. Ionic strength was relevant since at 150 mM NaCl, the process originating the endotherm at the highest temperature was irreversible (T(m2) = 108.5°C) with an activation energy of 703.8 kJ·mol(-1). At 500 mM NaCl the process became reversible (T(m2) = 114.4°C) and data were fitted to the Non-two States model with two subpeaks. No free thiols in the propeptide could be titrated by DTNB with or without 5.7 M GdmCl, indicating disulfide bonds establishment.