Effect of Detergents on Galactoside Binding by Melibiose Permeases.
The effect of various detergents on the stability and function of the melibiose permeases of Escherichia coli (MelBEc) and Salmonella typhimurium (MelBSt) was studied. In n-dodecyl-β-d-maltoside (DDM) or n-undecyl-β-d-maltoside (UDM), WT MelBSt binds melibiose with an affinity similar to that in the membrane. However, with WT MelBEc or MelBSt 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. MelBSt is more thermostable than MelBEc, 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 MelBSt 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.
Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center , Lubbock, Texas 79430, United States.
Anowarul
A
Amin
Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center , Lubbock, Texas 79430, United States.
Parameswaran
P
Hariharan
Department of Bionanotechnology, Hanyang University , Ansan 426-791, Korea.
Pil Seok
PS
Chae
Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center , Lubbock, Texas 79430, United States.
Lan
L
Guan
D003902:Detergents / Q000378:metabolism*; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005697:Galactosides / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism; D008553:Melibiose / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding / Q000187:drug effects; D012486:Salmonella typhimurium / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D027981:Symporters / Q000235:genetics / Q000378:metabolism*
D003902:Detergents; D005697:Galactosides; D005960:Glucosides; D027981:Symporters; C040358:dodecyl maltoside; C021548:melibiose permease; D008553:Melibiose