Bañares-Hidalgo Ángeles, Pérez-Gil Jesús, Estrada Pilar
Departamento de Bioquímica y Biología Molecular I, Facultad de Biología, Universidad Complutense, Ciudad Universitaria, 28040, Madrid, Spain.
PLoS One. 2016 Jul 5;11(7):e0158430. doi: 10.1371/journal.pone.0158430. eCollection 2016.
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-BN) 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-BN with trp fluorescence and circular dichroism spectroscopies. Binding of the intermediate states to bis-ANS suggests their molten globule-like character. ΔG0H2O was ~ 12.7 kJ·mol-1 either with urea or GdmCl. None of the thermal transitions of SP-BN detected by CD correspond to protein unfolding. Differential scanning calorimetry of SP-BN 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 (Tm2 = 108.5°C) with an activation energy of 703.8 kJ·mol-1. At 500 mM NaCl the process became reversible (Tm2 = 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.
肺表面活性物质脂质-蛋白质复合物的组装依赖于构象变化，这种变化与肺表面活性物质蛋白B（SP-B）的前体proSP-B在肺细胞表面活性物质生物合成途径中的蛋白水解成熟过程相关联。proSP-B的N端前肽（SP-BN）的构象不稳定会触发成熟SP-B结构域的暴露，以便插入表面活性物质脂质中。我们利用色氨酸荧光光谱和圆二色光谱研究了在GdmCl或尿素促进下SP-BN展开过程中的构象稳定性。中间态与双-ANS的结合表明它们具有类似熔球的性质。无论是使用尿素还是GdmCl，ΔG0H2O约为12.7 kJ·mol-1。通过圆二色光谱检测到的SP-BN的热转变均不对应于蛋白质的展开。SP-BN 的差示扫描量热法证明有两个吸热峰参与寡聚体解离，2 M尿素证实了这一点。离子强度很重要因为在150 mM NaCl时，在最高温度产生吸热峰的过程是不可逆的（Tm2 = 108.5°C），活化能为703.8 kJ·mol-1。在500 mM NaCl时，该过程变为可逆（Tm2 = 114.4°C），数据拟合到具有两个子峰的非二态模型。无论有无5.7 M GdmCl，前肽中均无游离巯基可被DTNB滴定滴定，表明二硫键已形成建立。
