The yeast mitochondrial citrate transport protein: determination of secondary structure and solvent accessibility of transmembrane domain IV using site-directed spin labeling.
The yeast mitochondrial citrate transport protein: determination of secondary structure and solvent accessibility of transmembrane domain IV using site-directed spin labeling.
复制标题
酵母线粒体柠檬酸转运蛋白：使用定点自旋标记确定跨膜结构域 IV 的二级结构和溶剂可及性。
DOI：
10.1021/bi000433e
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发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
McHaourab,HS
中科院分区：
文献类型：
--
作者：
Kaplan,RS;Mayor,JA;Kotaria,R;Walters,DE;McHaourab,HS
为了探索柠檬酸盐转运蛋白（CTP）的空间组织和功能动力学，沿着第四跨膜结构域（TMDIV）内的沿着22个连续残基进行氮氧化物扫描。该结构域被认为对CTP机制具有独特的重要性，这是由于（i）存在CTP功能所必需的两个膜内正电荷和（ii）在该结构域内存在跨膜水表面，其可能对应于柠檬酸盐易位途径的一部分。引入的氮氧自由基的迁移率的序列特异性变化及其对分子O2的辅助作用揭示了沿序列的α-螺旋构象沿着。的accessibilities NiEDDA是与accessibilites O2的阶段，表明一个面的螺旋是由脂双层溶剂化，而另一个是溶剂化的水性环境。沿着面对水相的螺旋表面观察到NiEDDA可及性的梯度，并且在这些位点处的EPR谱线形状指示相当大的运动限制。在模型的上下文中，TMDIV线的易位途径，这些数据表明通过该途径的被动扩散的障碍。本文首次报道了利用定点自旋标记技术研究线粒体转运蛋白的结构。
To explore the spatial organization and functional dynamics of the citrate transport protein (CTP), a nitroxide scan was carried out along 22 consecutive residues within the fourth transmembrane domain (TMDIV). This domain has been implicated as being of unique importance to the CTP mechanism due to (i) the presence of two intramembranous positive charges that are essential for CTP function and (ii) the existence of a transmembrane aqueous surface within this domain which likely corresponds to a portion of the citrate translocation pathway. The sequence-specific variation in the mobilities of the introduced nitroxides and their accessibilities to molecular O2reveal an α-helical conformation along the sequence. The accessibilities to NiEDDA are out of phase with accessibilites to O2, indicating that one face of the helix is solvated by the lipid bilayer while the other is solvated by an aqueous environment. A gradient of NiEDDA accessibility is observed along the helix surface facing the aqueous phase, and the EPR spectral line shapes at these sites indicate considerable motional restriction. In the context of the model where TMDIV lines the translocation pathway, these data suggest a barrier to passive diffusion through the pathway. This paper reports the first use of site-directed spin labeling to study mitochondrial transporter structure.