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

¹ Department of Biochemistry and Biophysical Chemistry, Groningen Biomolecular Science and Biotechnology Institute & Materials Science Centre^plus, University of Groningen, 9747 AG Groningen, The Netherlands
² MicroSpectroscopy Centre, Wageningen University, 6700 ET Wageningen, The Netherlands

(RECEIVED January 25, 2006; FINAL REVISION April 11, 2006; ACCEPTED April 14, 2006)

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.

Keywords: EnzymeII; mannitol transporter; fluorescence correlation spectroscopy; oligomeric state; oligomeric stability

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