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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Probing of the rates of alternating access in LacY with Trp fluorescence
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Probing of the rates of alternating access in LacY with Trp fluorescence

机译:用Trp荧光探测LacY中的交替访问速率

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Sugar/H~+ symport by lactose permease (LacY) utilizes an alternating access mechanism in which sugar and H~+ binding sites in the middle of the molecule are alternatively exposed to either side of the membrane by sequential opening and closing of inward- and outward-facing hydrophilic cavities. Here, we introduce Trp residues on either side of LacY where they are predicted to be in close proximity to side chains of natural Trp quenchers in either the inward- or outward-facing conformers. In the inward-facing con-former, LacY is tightly packed on the periplasmic side, and Trp residues placed at positions 245 (helix VII) or 378 (helix XII) are in close contact with His-35 (helix I) or Lys-42 (helix II), respectively. Sugar binding leads to unquenching of Trp fluorescence in both mutants, a finding clearly consistent with opening of the periplasmic cavity. The pH dependence of Trp-245 unquenching exhibits a pK_a of 8, typical for a His side chain interacting with an aromatic group. As estimated from stopped-flow studies, the rate of sugar-induced opening is ≈100 s~(-1). On the cytoplasmic side, Phe-140 (helix V) and Phe-334 (helix X) are located on opposite sides of a wide-open hydrophilic cavity. In precisely the opposite fashion from the periplasmic side, mutant Phe-140→Trp/Phe-334→His exhibits sugar-induced Trp quenching. Again, quenching is pH dependent (pK_a = 8), but remarkably, the rate of sugar-induced quenching is only ≈0.4 s~(-1). The results provide yet another strong, independent line of evidence for the alternating access mechanism and demonstrate that the methodology described provides a sensitive probe to measure rates of conformational change in membrane transport proteins.
机译:乳糖通透酶(LacY)的糖/ H〜+共存利用交替的进入机制,其中分子中部的糖和H〜+结合位点通过依次打开和关闭内向和向内暴露于膜的任一侧。向外的亲水腔。在这里,我们在LacY的任一侧引入Trp残基,预计它们在向内或向外构象异构体中都与天然Trp淬灭剂的侧链非常接近。在面向内的构象异构体中,LacY紧密堆积在周质侧,位于245(螺旋VII)或378(螺旋XII)位置的Trp残基与His-35(螺旋I)或Lys-紧密接触。 42(螺旋线II)。糖结合导致两个突变体中Trp荧光的猝灭,这一发现显然与周质腔的开放一致。 Trp-245淬灭的pH依赖性显示pK_a为8,通常用于His侧链与芳香族基团相互作用。根据停流研究的估计,糖诱导的开放速率约为100 s〜(-1)。在细胞质一侧,Phe-140(螺旋V)和Phe-334(螺旋X)位于一个敞开的亲水腔的相对两侧。与周质相反,突变体Phe-140→Trp / Phe-334→His具有糖诱导的Trp淬灭作用。同样,淬灭是pH依赖性的(pK_a = 8),但值得注意的是,糖诱导的淬灭速率仅为≈0.4s〜(-1)。结果为交替进入机制提供了又一个强有力的独立证据,并证明所描述的方法提供了灵敏的探针来测量膜转运蛋白的构象变化率。

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