Site directed spin-labeling (SDSL) has been used to probe the structural and dynamic features of residues comprising the sixth transmembrane segment of the mitochondrial oxoglutarate carrier. Starting from a functional carrier, where cysteines have been replaced by serines, 18 consecutive residues (from G281 to I298) have been mutated to cysteine and subsequently labeled with a thiol-selective nitroxide probe. The labeled proteins, reconstituted into liposomes, have been assayed for their transport activity and analyzed with continuous-wave electron paramagnetic resonance. Linewidth analysis, that is correlated to local probe mobility, indicates a well defined periodicity of the whole segment from G281 to I298, indicating that it has an alpha-helical structure. Saturation behaviour, in presence of paramagnetic perturbants of different hydrophobicities, allow the definition of the polarity of the individual residues and to assign their orientation with respect to the lipid bilayer or to the water accessible translocation channel. Comparison of the EPR data, homology model and activity data indicate that the segment is made by an alpha helix, accommodated in an amphipathic environment, partially distorted in the middle at the level of L289, probably because of the presence of a proline residue (P291). The C-terminal region of the segment is less restrained and more flexible than the N-terminus.

中文翻译：

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通过定点自旋标记研究线粒体氧戊二酸酯载体的跨膜区段VI的结构动力学特性。

定点自旋标记（SDSL）已用于探测包含线粒体氧戊二酸酯载体的第六个跨膜片段的残基的结构和动态特征。从功能性载体开始，其中半胱氨酸已被丝氨酸取代，已将18个连续残基（从G281到I298）突变为半胱氨酸，随后用硫醇选择性氮氧化物探针标记。已经对重构为脂质体的标记蛋白质的转运活性进行了测定，并通过连续波电子顺磁共振进行了分析。与局部探针迁移率相关的线宽分析表明，从G281到I298的整个片段的周期定义明确，表明它具有α-螺旋结构。饱和行为，在存在不同疏水性的顺磁性扰动剂的情况下，可以定义各个残基的极性，并指定其相对于脂质双层或水易位通道的方向。EPR数据，同源性模型和活性数据的比较表明，该片段由α螺旋组成，位于两亲环境中，在L289水平的中间部分扭曲，可能是由于脯氨酸残基的存在（P291 ）。该片段的C末端区域比N末端约束更少，更灵活。同源性模型和活性数据表明，该片段由容纳在两亲性环境中的α螺旋组成，在L289的水平中间部分扭曲，这可能是由于脯氨酸残基（P291）的存在。该片段的C末端区域比N末端约束更少，更灵活。同源性模型和活性数据表明，该片段由容纳在两亲性环境中的α螺旋组成，在L289的水平中间部分扭曲，这可能是由于脯氨酸残基（P291）的存在。该片段的C末端区域比N末端约束更少，更灵活。