Effect of Detergents on Galactoside Binding by Melibiose Permeases

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• 作者：Anowarul Amin ; Parameswaran Hariharan ; Pil Seok Chae ; Lan Guan
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：September 29, 2015
• 年：2015
• 卷：54
• 期：38
• 页码：5849-5855
• 全文大小：386K
• ISSN：1520-4995

文摘

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-尾-d-maltoside (DDM) or n-undecyl-尾-d-maltoside (UDM), WT MelB_St binds melibiose with an affinity similar to that in the membrane. However, with WT MelB_Ec or MelB_St mutants (Arg141 鈫?Cys, Arg295 鈫?Cys, or Arg363 鈫?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 thermostable 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 calorimetry 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.