Frillingos S, Sahin-Tóth M, Wu J, Kaback H R
Howard Hughes Medical Institute, Departments of Physiology and Microbiology and Molecular Genetics, Molecular Biology Institute, University of California Los Angeles, Los Angeles, California 90024
FASEB J. 1998 Oct;12(13):1281-99. doi: 10.1096/fasebj.12.13.1281.
The entire lactose permease of Escherichia coli, a polytopic membrane transport protein that catalyzes beta-galactoside/H+ symport, has been subjected to Cys-scanning mutagenesis in order to determine which residues play an obligatory role in the mechanism and to create a library of mutants with a single-Cys residue at each position of the molecule for structure/function studies. Analysis of the mutants has led to the following: 1) only six amino acid side chains play an irreplaceable role in the transport mechanism; 2) positions where the reactivity of the Cys replacement is increased upon ligand binding are identified; 3) positions where the reactivity of the Cys replacement is decreased by ligand binding are identified; 4) helix packing, helix tilt, and ligand-induced conformational changes are determined by using the library of mutants in conjunction with a battery of site-directed techniques; 5) the permease is a highly flexible molecule; and 6) a working model that explains coupling between beta-galactoside and H+ translocation. structure-function relationships in polytopic membrane proteins.
大肠杆菌的整个乳糖通透酶是一种催化β-半乳糖苷/H⁺同向转运的多跨膜转运蛋白，已对其进行了半胱氨酸扫描诱变，以确定哪些残基在该机制中起关键作用，并创建一个在分子每个位置都有单个半胱氨酸残基的突变体库用于结构/功能研究。对这些突变体的分析得出了以下结果：1）只有六个氨基酸侧链在转运机制中起不可替代的作用；2）确定了半胱氨酸替代物在配体结合后反应性增加的位置；3）确定了半胱氨酸替代物在配体结合后反应性降低的位置；4）通过将突变体库与一系列定点技术结合使用，确定了螺旋堆积、螺旋倾斜和配体诱导的构象变化；5）通透酶是一种高度灵活的分子；6）一个解释β-半乳糖苷与H⁺转运之间偶联的工作模型。多跨膜蛋白中的结构-功能关系。
