Oligomeric state of membrane transport proteins analyzed with blue native electrophoresis and analytical ultracentrifugation.
Blue native electrophoresis is used widely for the analysis of non-dissociated protein complexes with respect to composition, oligomeric state and molecular mass. However, the effects of detergent or dye binding on the mass and stability of the integral membrane proteins have not been studied. By comparison with analytical ultracentrifugation, we have evaluated whether the oligomeric state of membrane transport proteins is reflected reliably with blue native electrophoresis. For the analysis we have used two well-characterized transporters, that is, the major facilitator superfamily protein LacS and the phosphotransferase system EII(Mtl). For another member of the major facilitator superfamily, the xyloside transporter XylP from Lactobacillus pentosus, the complete analysis of the quaternary structure determined by analytical ultracentrifugation and freeze-fracture electron microscopy is presented. Our experiments show that during blue native electrophoresis the detergent bound to the proteins is replaced by the amphipathic Coomassie brilliant blue (CBB) dye. The mass of the bound CBB dye was quantified. Provided this additional mass of bound CBB dye is accounted for and care is taken in the choice and concentration of the detergent used, the mass of LacS, XylP and EII(Mtl) and four other membrane (transport) proteins could be deduced within 10 % error. Our data underscore the fact that the oligomeric state of many membrane transport proteins is dimeric.
Heuberger
Esther H M L
EH
Department of Biochemistry Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, the Netherlands.
Veenhoff
Liesbeth M
LM
Duurkens
Ria H
RH
Friesen
Robert H E
RH
Poolman
Bert
B
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D002138:Calibration; D003902:Detergents / Q000494:pharmacology; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry; D005614:Freeze Fracturing; D013294:Lactococcus lactis / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D008970:Molecular Weight; D020836:Protein Structure, Quaternary / Q000187:drug effects; D012995:Solubility / Q000187:drug effects; D016963:Thermus thermophilus / Q000737:chemistry; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D003902:Detergents; D026901:Membrane Transport Proteins