Structure/function relationships in OxlT, the oxalate/formate antiporter of Oxalobacter formigenes: assignment of transmembrane helix 2 to the translocation pathway.
Structure/function relationships in OxlT, the oxalate/formate antiporter of Oxalobacter formigenes: assignment of transmembrane helix 2 to the translocation pathway.
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OxlT（产酸草杆菌的草酸盐/甲酸盐逆向转运蛋白）的结构/功能关系：跨膜螺旋 2 分配给易位途径。
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发表时间：
2002
期刊：
影响因子：
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通讯作者：
P. Maloney
中科院分区：
文献类型：
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作者：
L. Ye;P. Maloney
我们构建了一个单一的半胱氨酸面板，包括跨膜螺旋2（TM 2）的OxlT，草酸/甲酸反向转运蛋白的Oxaltophorformigenes。在21个目标位置中，半胱氨酸取代鉴定出一个（苯丙氨酸59）对OxlT表达是必需的，三个（谷氨酰胺56、谷氨酰胺66和丝氨酸69）对OxlT功能是潜在关键的。通过用大体积的亲水性探针（俄勒冈州绿色马来酰亚胺）探测膜，我们还定位了一个中央不可接近的核心，长度至少为8个残基，从亮氨酸61延伸到甘氨酸68。基于粗洗涤剂提取物重构的功能测定显示，在TM 2核心内的单个半胱氨酸突变体中，只有Q63 C变体基本上（>或=95%）被硫醇特异性试剂（羧乙基甲硫代磺酸盐和乙基磺酸盐甲硫代磺酸盐）抑制。随后的分析工作，使用纯化的Q63 C蛋白质表明，抑制由乙磺酸甲硫基磺酸盐被阻断的基板和浓度依赖性的这种基板保护发生与结合常数为0.16毫米草酸盐，与米氏常数观察到的草酸盐运输（0.23毫米）。这些发现使我们得出结论，位置63位于OxlT易位途径上。我们的结论得到以下发现的加强：位置63，沿着与TM 2功能相关的大多数其他位置，位于可以与TM 11的面向通路的表面交联的螺旋面上（Fu，D.，萨克河一、博尔顿，和Maloney，P. C.（2001）J.Biol.Chem.276，8753-8760）。
We constructed a single cysteine panel encompassing transmembrane helix two (TM2) of OxlT, the oxalate/formate antiporter of Oxalobacter formigenes. Among the 21 positions targeted, cysteine substitution identified one (phenylalanine 59) as essential to OxlT expression and three (glutamine 56, glutamine 66, and serine 69) as potentially critical to OxlT function. By probing membranes with a bulky hydrophilic probe (Oregon Green maleimide) we also located a central inaccessible core of at least eight residues in length, extending from leucine 61 to glycine 68. Functional assays based on reconstitution of crude detergent extracts showed that of single cysteine mutants within the TM2 core only the Q63C variant was substantially (> or =95%) inhibited by thiol-specific agents (carboxyethyl methanethiosulfonate and ethylsulfonate methanethiosulfonate). Subsequent analytical work using the purified Q63C protein showed that inhibition by ethylsulfonate methanethiosulfonate was blocked by substrate and that the concentration dependence of such substrate protection occurred with a binding constant of 0.16 mm oxalate, comparable with the Michaelis constant observed for oxalate transport (0.23 mm). These findings lead us to conclude that position 63 lies on the OxlT translocation pathway. Our conclusion is strengthened by the finding that position 63, along with most other positions relevant to TM2 function, is found on a helical face that can be cross-linked to the pathway-facing surface of TM11 (Fu, D., Sarker, R. I., Bolton, E., and Maloney, P. C. (2001) J. Biol. Chem. 276, 8753-8760).