The oligomeric state and stability of the mannitol transporter, EnzymeII(mtl), from Escherichia coli: a fluorescence correlation spectroscopy study.
Numerous membrane proteins function as oligomers both at the structural and functional levels. The mannitol transporter from Escherichia coli, EnzymeII(mtl), is a member of the phosphoenolpyruvate-dependent phosphotransferase system. During the transport cycle, mannitol is phosphorylated and released into the cytoplasm as mannitol-1-phosphate. Several studies have shown that EII(mtl) functions as an oligomeric species. However, the oligomerization number and stability of the oligomeric complex during different steps of the catalytic cycle, e.g., substrate binding and/or phosphorylation of the carrier, is still under discussion. In this paper, we have addressed the oligomeric state and stability of EII(mtl) using fluorescence correlation spectroscopy. A functional double-cysteine mutant was site-specifically labeled with either Alexa Fluor 488 or Alexa Fluor 633. The subunit exchange of these two batches of proteins was followed in time during different steps of the catalytic cycle. The most important conclusions are that (1) in a detergent-solubilized state, EII(mtl) is functional as a very stable dimer; (2) the stability of the complex can be manipulated by changing the intermicellar attractive forces between PEG-based detergent micelles; (3) substrate binding destabilizes the complex whereas phosphorylation increases the stability; and (4) substrate binding to the phosphorylated species partly antagonizes the stabilizing effect.
Veldhuis
Gertjan
G
Department of Biochemistry and Biophysical Chemistry, Groningen Biomolecular Science and Biotechnology Institute & Materials Science Centreplus, University of Groningen, The Netherlands.
Hink
Mark
M
Krasnikov
Victor
V
van den Bogaart
Geert
G
Hoeboer
Jeroen
J
Visser
Antonie J W G
AJ
Broos
Jaap
J
Poolman
Bert
B
D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D018613:Microscopy, Confocal; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000235:genetics; D010766:Phosphorylation; D020836:Protein Structure, Quaternary; D013050:Spectrometry, Fluorescence
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli; D003545:Cysteine