Oligomeric state of membrane transport proteins analyzed with blue native electrophoresis and analytical ultracentrifugation
Oligomeric state of membrane transport proteins analyzed with blue native electrophoresis and analytical ultracentrifugation
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DOI：
10.1006/jmbi.2002.5416
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发表时间：
2002-04-05
影响因子：
5.6
通讯作者：
Poolman, B
中科院分区：
文献类型：
--
作者：
Heuberger, EHML;Veenhoff, LM;Poolman, B
蓝色原生电泳广泛用于分析非解离蛋白复合物的组成、寡聚态和分子质量。然而，洗涤剂或染料结合对整体膜蛋白质量和稳定性的影响尚未研究。通过与分析性超离心的比较，我们评估了蓝色原生电泳是否可靠地反映了膜运输蛋白的低聚状态。为了进行分析，我们使用了两种特性良好的转运蛋白，即主要的促进剂超家族蛋白LacS和磷酸转移酶系统EIIMtl。对于主要促进剂超家族的另一个成员，来自戊酸乳杆菌的木糖苷转运体XylP，通过分析超离心和冷冻断裂电镜对其进行了完整的四级结构分析。我们的实验表明，在蓝色原生电泳过程中，与蛋白质结合的洗涤剂被两亲性考马斯亮蓝（CBB）染料所取代。测定了结合的CBB染料的质量。如果考虑到这一额外的结合CBB的质量，并且注意洗涤剂的选择和浓度，那么LacS、XylP和EIIMtl以及其他四种膜（运输）蛋白的质量可以在10%的误差内推断出来。我们的数据强调了这样一个事实，即许多膜转运蛋白的低聚状态是二聚体。(C) 2002 Elsevier Science Ltd.
Blue native electrophoresis is used widely for the analysis of non-dissociated protein complexes with respect to composition, oligomeric state and molecular mass. However, the effects of detergent or dye binding on the mass and stability of the integral membrane proteins cave not been studied. By comparison with analytical ultracentrifugation, we have evaluated whether the oligomeric state of membrane transport proteins is reflected reliably with blue native electrophoresis. For the analysis we have used two well-characterized transporters, that is, the major facilitator superfamily protein LacS and the phosphotransferase system EIIMtl. For another member of the major facilitator superfamily, the xyloside transporter XylP from Lactobacillus pentosus, the complete analysis of the quaternary structure determined by analytical ultracentrifugation and freeze-fracture electron microscopy is presented.Our experiments show that during blue native electrophoresis the detergent bound to the proteins is replaced by the amphipathic Coomassie brilliant blue (CBB) dye. The mass of the bound CBB dye was quantified. Provided this additional mass of bound CBB dve is accounted for and care is taken in the choice and concentration of the detergent used, the mass of LacS, XylP and EIIMtl and four other membrane (transport) proteins could be deduced within 10% error. Our data underscore the fact that the oligomeric state of many membrane transport proteins is dimeric. (C) 2002 Elsevier Science Ltd.