Nie Yiling, Kaback H Ronald
Department of Physiology, Department of Microbiology, Immunology & Molecular Genetics, and Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.
Proc Natl Acad Sci U S A. 2010 May 25;107(21):9903-8. doi: 10.1073/pnas.1004515107. Epub 2010 May 10.
Many independent lines of evidence indicate that the lactose permease of Escherichia coli (LacY) is highly dynamic and that sugar binding causes closing of a large inward-facing cavity with opening of a wide outward-facing hydrophilic cavity. Therefore, lactose/H(+) symport catalyzed by LacY very likely involves a global conformational change that allows alternating access of single sugar- and H(+)-binding sites to either side of the membrane (the alternating access model). The x-ray crystal structures of LacY, as well as the majority of spectroscopic studies, use purified protein in detergent micelles. By using site-directed alkylation, we now demonstrate that sugar binding induces virtually the same global conformational change in LacY whether the protein is in the native bacterial membrane or is solubilized and purified in detergent. The results also indicate that the x-ray crystal structure reflects the structure of wild-type LacY in the native membrane in the absence of sugar.
许多独立的证据表明，大肠杆菌乳糖通透酶（LacY）具有高度动态性，并且糖结合会导致一个大的向内开放腔关闭，同时一个宽阔的向外开放亲水腔打开。因此，LacY催化的乳糖/H(+)同向转运很可能涉及一种全局构象变化，这种变化允许单个糖结合位点和H(+)结合位点交替与膜的两侧接触（交替访问模型）。LacY的X射线晶体结构以及大多数光谱研究，都是使用在去污剂胶束中的纯化蛋白进行的。通过定点烷基化，我们现在证明，无论蛋白质是处于天然细菌膜中，还是在去污剂中溶解和纯化，糖结合都会在LacY中诱导几乎相同的全局构象变化。结果还表明，X射线晶体结构反映了野生型LacY在天然膜中无糖状态下的结构。
