Ujwal M L, Jung H, Bibi E, Manoil C, Altenbach C, Hubbell W L, Kaback H R
Howard Hughes Medical Institute, Department of Physiology, Los Angeles, California, USA.
Biochemistry. 1995 Nov 14;34(45):14909-17. doi: 10.1021/bi00045a036.
The use of lactose permease-alkaline phosphatase fusions (lacY-phoA) demonstrates that the lactose permease of Escherichia coli contains 12 transmembrane domains and that approximately half of a transmembrane domain is required to translocate alkaline phosphatase to the periplasmic surface of the membrane [Calamia, J., & Manoil, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4937-4941]. We have now used fusion analysis in combination with site-directed spectroscopy to examine more precisely the topology of putative helices VII and XI which contain the interacting residues Asp237 and Lys358, respectively. For this purpose, alkaline phosphatase was fused to alternate amino acid residues in transmembrane domains VII and XI. A sharp increase in alkaline phosphatase activity is observed as the fusion junction proceeds from Try228 to Ile230 in helix VII and from Phe354 to Phe356 in helix XI, suggesting that these residues approximate the middle of the corresponding transmembrane helices. Analysis of fluorescence quenching of the pyrene-labeled single-Cys mutants Asp237 --> Cys or Lys358 --> Cys, as well as measurement of collision frequencies between freely diffusing paramagnetic probes and a nitroxide spin-label at these sites, also indicates that Asp237 and also Asp240, which interacts with Lys319 (helix X), are located in transmembrane domains. However, Asp237 and Asp240 are accessible both from the aqueous phase and from within the membrane. The results provide more direct evidence that the three residues are located within transmembrane helices and suggest that Asp237 and Asp240 are either located near the periplasmic surface of the membrane or exposed within a solvent-filled cleft in the permease.
乳糖通透酶-碱性磷酸酶融合蛋白（lacY-phoA）的应用表明，大肠杆菌的乳糖通透酶含有12个跨膜结构域，并且大约半个跨膜结构域就足以将碱性磷酸酶转运到膜的周质表面[卡拉米亚，J.，& 马诺伊尔，C.（1990年）《美国国家科学院院刊》87卷，4937 - 4941页]。我们现在将融合分析与定点光谱法相结合，更精确地研究推定的螺旋VII和XI的拓扑结构，这两个螺旋分别包含相互作用的残基Asp237和Lys358。为此，将碱性磷酸酶与跨膜结构域VII和XI中的交替氨基酸残基融合。当融合连接点从螺旋VII中的Try228延伸至Ile230以及从螺旋XI中的Phe354延伸至Phe356时，观察到碱性磷酸酶活性急剧增加，这表明这些残基接近相应跨膜螺旋的中部。对芘标记的单半胱氨酸突变体Asp237→Cys或Lys358→Cys的荧光猝灭分析，以及对自由扩散的顺磁探针与这些位点的氮氧化物自旋标记之间碰撞频率的测量，也表明与Lys319（螺旋X）相互作用的Asp237以及Asp240位于跨膜结构域中。然而，Asp237和Asp240既可以从水相进入，也可以从膜内部进入。这些结果提供了更直接的证据，证明这三个残基位于跨膜螺旋内，并表明Asp237和Asp240要么位于膜的周质表面附近，要么暴露于通透酶中充满溶剂的裂隙内。
