Ding Ping Z
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
Biochem Biophys Res Commun. 2003 Aug 8;307(4):864-9. doi: 10.1016/s0006-291x(03)01290-7.
The melibiose transporter (Mel B) of Escherichia coli is a cation-coupled (H(+), Li(+), and Na(+)) membrane protein (MW 50 kDa) consisting of 12 transmembrane helices that are connected by periplasmic and cytoplasmic loops, with both the C- and N-ends located on the cytoplasmic side of the membrane. Previous investigations on the largest cytoplasmic loop X/XI indicated that it is a functional re-entrant loop. In this communication, the cysteine mutants on loop X/XI were studied with charged thiol reagents MTSES, MTSET, and IAA for both the inhibition patterns and charge replacement/function rescue of inactive mutants in which the original charged residues were replaced by neutral cysteines. Strong inhibitions were observed in T373C and V376C by both MTSES and MTSET, consistent with previous results of PCMBS inhibition. The thiol reagents failed to recover the activities of inactive mutants D351C, D354C, and R363C and to inhibit active mutants E357C, K359C, and E365C to any significant extent, suggesting a structural conservation at D351, D354, and R363 and tolerance of structural variations at E357, K359, and E365. The results are consistent with previous observation of structural conservation of functionally charged residues in the transmembrane domains and extend to a loop the contention that in the melibiose transporter functionally important charged residues are structurally conserved.
大肠杆菌的蜜二糖转运蛋白（Mel B）是一种阳离子偶联（H⁺、Li⁺和Na⁺）膜蛋白（分子量50 kDa），由12个跨膜螺旋组成，这些跨膜螺旋通过周质环和细胞质环相连，C端和N端均位于膜的细胞质一侧。先前对最大的细胞质环X/XI的研究表明，它是一个功能性折返环。在本通讯中，使用带电硫醇试剂MTSES、MTSET和IAA研究了环X/XI上的半胱氨酸突变体的抑制模式以及原始带电残基被中性半胱氨酸取代的无活性突变体的电荷置换/功能挽救情况。MTSES和MTSET在T373C和V376C中均观察到强烈抑制，这与之前PCMBS抑制的结果一致。硫醇试剂未能恢复无活性突变体D351C、D354C和R363C的活性，也未能在任何显著程度上抑制活性突变体E357C、K359C和E365C，这表明D351、D354和R363处存在结构保守性，而E357、K359和E365处对结构变化具有耐受性。这些结果与之前在跨膜结构域中对功能性带电残基结构保守性的观察结果一致，并将在蜜二糖转运蛋白中功能性重要带电残基在结构上保守的观点扩展到了一个环上。
