Effect of Detergents on Galactoside Binding by Melibiose Permeases
Effect of Detergents on Galactoside Binding by Melibiose Permeases
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DOI：
10.1021/acs.biochem.5b00660
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发表时间：
2015-09-29
期刊：
影响因子：
2.9
通讯作者：
Guan, Lan
中科院分区：
文献类型：
--
作者：
Amin, Anowarul;Hariharan, Parameswaran;Guan, Lan
研究了不同去污剂对大肠杆菌（MelB（Ec））和鼠伤寒沙门氏菌（MelB（st））蜜二糖渗透酶稳定性和功能的影响。在正十二烷基-β-D-麦芽糖苷（DDM）或正十一烷基-β-D-麦芽糖苷（UDM）中，WT MelBst以与膜中相似的亲和力结合蜜二糖。然而，对于WT MelB（Ec）或MelB（st）突变体（Arg 141-> Cys、Arg 295-> Cys或Arg 363-> Cys），在这些去污剂中未检测到半乳糖苷结合，但保持与磷酸转移酶蛋白IIA（Glc）的结合。在两亲物月桂基麦芽糖新戊二醇（MNG-3）或glyco-diosgenin（GDN）中，观察到半乳糖苷与所有MelB蛋白结合，亲和力略有降低。MelB（st）比MelB（Ec）更热稳定，并且MelB的热稳定性在MNG-3或GDN中大大增加。因此，DDM或UDM的功能缺陷可能是由敏感MelB蛋白的相对不稳定性引起的，并且稳定性以及半乳糖苷结合在MNG-3或GDN中得以保留。此外，MelB（st）与蜜二糖结合的等温滴定热分析表明，在MNG-3中，MelB的构象动力学受到限制，熵对结合自由能的贡献降低.
The effect of various detergents on the stability and function of the melibiose permeases of Escherichia coli (MelB(Ec)) and Salmonella typhimurium (MelB(st)) was studied. In n-dodecyl-beta-D-maltoside (DDM) or n-undecyl-beta-Dmaltoside (UDM), WT MelBst binds melibiose with an affinity similar to that in the membrane. However, with WT MelB(Ec), or MelB(st) mutants (Arg141 -> Cys, Arg295 -> Cys, or Arg363 -> Cys), galactoside binding is not detected in these detergents, but binding to the phosphotransferase protein IIA(Glc) is maintained. In the amphiphiles lauryl maltose neopentyl glycol (MNG-3) or glyco-diosgenin (GDN), galactoside binding with all of the MelB proteins is observed, with slightly reduced affinities. MelB(st) is more therrnostable than MelB(Ec) and the thermostability of either MelB is largely increased in MNG-3 or GDN. Therefore, the functional defect with DDM or UDM likely results from the relative instability of the sensitive MelB proteins, and stability, as well as galactoside binding, is retained in MNG-3 or GDN. Furthermore, isothermal titration calorirnetry of melibiose binding with MelB(st) shows that the favorable entropic contribution to the binding free energy is decreased in MNG-3, indicating that the conformational dynamics of MelB is restricted in this detergent.