The oligomeric state and stability of the mannitol transporter, EnzymeII super(mtl), from Escherichia coli: A fluorescence correlation spectroscopy study

• Published in: Protein science 2006-08, Vol.15 (8), p.1977-1986
• Main Authors: Veldhuis, Gertjan, Hink, Mark, Krasnikov, Victor, van den Bogaart, Geert, Hoeboer, Jeroen, Visser, Antonie JWG, Broos, Jaap, Poolman, Bert
• Format: Article
• Language: English
• Subjects: Escherichia coli
• ISSN: 0961-8368

Numerous membrane proteins function as oligomers both at the structural and functional levels. The mannitol transporter from Escherichia coli, EnzymeII super(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 super(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 super(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 super(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.