Hydrophobic core of the steroidogenic acute regulatory protein for cholesterol transport.
Hydrophobic core of the steroidogenic acute regulatory protein for cholesterol transport.
复制标题
胆固醇转运的类固醇生成急性调节蛋白的疏水核心。
DOI：
10.1021/bi801514e
复制
发表时间：
2009
期刊：
影响因子：
2.9
通讯作者：
Debnath,Dilip
中科院分区：
文献类型：
--
作者：
Bose,HimangshuS;Whittal,RandyM;Bose,Mahuya;Debnath,Dilip
类固醇合成急性调节蛋白(STAR)是STAR(STAR)蛋白家族的第一个成员，通过促进胆固醇从线粒体膜外到内膜的运动而发挥重要作用。野生型和突变型STAR以相似的强度结合胆固醇，但只有野生型STAR才能将其运输到线粒体。在这里，我们报告了疏水核心对具有起始结构域的蛋白质的生物活性至关重要。野生型STAR的类固醇合成活性是突变体R182L的7−9倍，但两者的近紫外圆二色谱相似。在相同尿素浓度下，野生型与突变型相比，其荧光最大值发生红移。TFE对野生型StAR的α-螺旋贡献大于突变蛋白。丙烯酰胺猝灭野生型蛋白(Ksv=12.0±0.2−11.2±0.5M−1)超过突变蛋白(Ksv=4±0.2M−1)。与这些发现一致的是，疏水探针与野生型恒星(KAPP=8.1×105M−1)的结合程度高于突变恒星(KAPP=3.75×105M−1)。质谱学分析表明，只有野生型STAR有一个对蛋白酶敏感的C末端，而突变体没有。停流CD显示突变星的展开时间为0.017 S，而野生型恒星蛋白的展开时间为16.3 S。综上所述，这些结果表明野生型恒星由于容纳了更多的水分子而采取了非常灵活的形式，而突变星是通过另一条折叠途径产生的，使其失去活性。
The steroidogenic acute regulatory protein (StAR), the first family member of START (StAR-related lipid transport) proteins, plays an essential role by facilitating the movement of cholesterol from the outer to inner mitochondrial membrane. Wild-type and mutant StAR binds cholesterol with similar intensity, but only wild-type StAR can transport it to mitochondria. Here, we report that the hydrophobic core is crucial for biological activity of proteins with START domains. Wild-type StAR increased steroidogenic activity by 7−9-fold compared to mutant R182L StAR, but both of them showed similar near-UV CD spectra. The fluorescence maximum of wild-type StAR is red shifted in comparison to mutant StAR under identical urea concentration. TFE increased the α-helical contribution of wild-type StAR more than the mutant protein. Acrylamide quenching for the wild-type protein (KSV= 12.0 ± 0.2−11.2 ± 0.5 M−1) exceeded that of the mutant protein (KSV= 4 ± 0.2 M−1). Consistent with these findings, the hydrophobic probe ANS bound wild-type StAR (Kapp= 8.1 × 105M−1) to a greater degree than mutant StAR (Kapp= 3.75 × 105M−1). Partial proteolysis examined by mass spectrometry suggests that only wild-type StAR has a protease-sensitive C-terminus, but not the mutant. Stopped-flow CD revealed that the time of unfolding of mutant StAR was 0.017 s. In contrast, the wild-type StAR protein is unfolded in 16.3 s. In summary, these results demonstrate that wild-type StAR adopts a very flexible form due to the accommodation of more water molecules, while mutant StAR is generated by an alternate folding pathway making it inactive.