Oligomeric state of membrane transport proteins analyzed with blue native electrophoresis and analytical ultracentrifugation

E.H M L Heuberger, L.M. Veenhoff, R.H.T. Duurkens, R.H.E. Friesen, B. Poolman

Research output: Contribution to journalArticleAcademicpeer-review

127 Citations (Scopus)
43 Downloads (Pure)

Abstract

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 EIIMtl. 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 EIIMtl 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.
Original languageDutch
Pages (from-to)591-600
Number of pages10
JournalJournal of Molecular Biology
Volume317
Issue number4
DOIs
Publication statusPublished - 5-Apr-2002

Keywords

  • freeze fracture electron microscopy
  • analytical ultracentrifugation
  • blue native electrophoresis
  • quaternary structure
  • membrane transport proteins

Cite this