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首页> 外文期刊>Biochemistry >Determining the membrane topology of proteins: insertion pathway of a transmembrane helix of annexin 12.
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Determining the membrane topology of proteins: insertion pathway of a transmembrane helix of annexin 12.

机译：确定蛋白质的膜拓扑：膜联蛋白12的跨膜螺旋的插入途径。

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We describe a sensitive method for determining the bilayer topology of single-site cysteine-linked NBD fluorescent labels on membrane proteins. Based upon a method developed for peptides [W. C. Wimley and S. H. White (2000) Biochemistry 39, 161-170], it utilizes a novel fluorescence quencher, lysoUB, comprised of a single acyl chain attached to a UniBlue chromophore. The enhanced sensitivity of the method arises from the brightness of the NBD fluorescence and the quenching efficiency of lysoUB, which is not fluorescent. In the course of validating the method, we examined the insertion topology of the D-E helical region of repeat 2 of annexin 12, known to adopt a transbilayer orientation at mildly acidic pH [Langen et al. (1998) Proc. Natl. Acad. Sci. USA 95, 14060-14065]. In the final membrane-inserted state, an NBD label attached to the single-cysteine mutant D134C was found to be in the outer (cis) leaflet, while the one attached to D162C was found in the trans leaflet. But kinetic measurements of NBD fluorescence suggested the existence of a transient intermediate insertion state whose lifetime could be increased by increasing the fraction of anionic lipids in the vesicles. Indeed, the lifetime could be increased for times sufficient for the completion of lysoUB-NBD topology measurements. Such measurements revealed that the D-E region adopts an interfacial topology in the intermediate state with both ends on the cis side of the membrane, consistent with the general concept of interface-directed membrane insertion of proteins [White et al. (2001) J. Biol. Chem. 276, 32395-32398].
机译：我们描述了一种确定单点半胱氨酸连接的NBD荧光标记在膜蛋白上的双层拓扑结构的灵敏方法。基于针对肽开发的方法[W. C.Wimley和S.H.White（2000）Biochemistry 39，161-170]，它利用了新型的荧光猝灭剂lysoUB，其由连接到UniBlue发色团的单个酰基链组成。该方法灵敏度的提高归因于NBD荧光的亮度和非荧光的lysoUB的猝灭效率。在验证该方法的过程中，我们检查了膜联蛋白12重复序列2的D-E螺旋区的插入拓扑结构，已知该结构在轻度酸性pH值下会采用跨双分子层取向[Langen等，2003年。 （1998）美国国家科学院院刊。 Natl。学院科学USA 95，14060-14065]。在最终的膜插入状态下，发现附着于单半胱氨酸突变体D134C的NBD标签位于外（顺式）小叶中，而附着于D162C的NBD标签位于反式小叶中。但是NBD荧光的动力学测量表明存在暂时的中间插入状态，其寿命可以通过增加囊泡中阴离子脂质的比例来延长。实际上，寿命可以延长足以完成lysoUB-NBD拓扑测量的时间。这样的测量表明，D-E区域在中间状态采用界面拓扑，其两端在膜的顺式侧，这与蛋白质的界面定向膜插入的一般概念一致[White等人。 （2001）J.Biol。化学276，32395-32398]。

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