Sahlman L, Hägglöf E M, Powlowski J
Department of Biochemistry, University of Umeå, Sweden.
Biochem Biophys Res Commun. 1999 Feb 16;255(2):307-11. doi: 10.1006/bbrc.1999.0192.
The roles of the four cysteine residues of the integral inner membrane Hg2+-binding protein, MerC, have been examined using site-directed mutagenesis. Residues Cys-22 and Cys-25 have previously been predicted to lie within the membrane. Substitution of each of these residues in turn with alanine resulted in complete abolition of specific Hg2+ uptake by vesicles. In contrast, substitution by alanine of the other two cysteine residues, Cys-127 and Cys-132, predicted to lie with within a C-terminal cytoplasmic tail, did not significantly affect Hg2+ uptake. Since previous results indicated that native MerC tends to form intermolecular disulfide-bonded dimers, the effects of these substitutions on dimer formation were also examined. Only the Cys-127 and Cys-132 variants spontaneously formed significant amounts of disulfide-bonded dimer. Further experiments using copper-1,10-phenanthroline indicated that each variant with an unpaired cysteine residue was more susceptible to dimer formation than native MerC.
利用定点诱变技术研究了内膜整合型汞结合蛋白MerC的4个半胱氨酸残基的作用。先前预测半胱氨酸-22和半胱氨酸-25残基位于膜内。依次用丙氨酸取代这些残基中的每一个，导致囊泡对特定汞离子的摄取完全丧失。相比之下，预测位于C端胞质尾部的另外两个半胱氨酸残基，即半胱氨酸-127和半胱氨酸-132，用丙氨酸取代后对汞离子摄取没有显著影响。由于先前的结果表明天然MerC倾向于形成分子间二硫键连接的二聚体，因此还研究了这些取代对二聚体形成的影响。只有半胱氨酸-127和半胱氨酸-132变体自发形成了大量二硫键连接的二聚体。使用铜-1,10-菲咯啉的进一步实验表明，每个带有未配对半胱氨酸残基的变体比天然MerC更容易形成二聚体。
