PMID:21935376
| Citation |
Aboulwafa, M and Saier, MH Jr (2011) Biophysical studies of the membrane-embedded and cytoplasmic forms of the glucose-specific Enzyme II of the E. coli phosphotransferase system (PTS). PLoS ONE 6:e24088 |
|---|---|
| Abstract |
The glucose Enzyme II transporter complex of the Escherichia coli phosphotransferase system (PTS) exists in at least two physically distinct forms: a membrane-integrated dimeric form, and a cytoplasmic monomeric form, but little is known about the physical states of these enzyme forms. Six approaches were used to evaluate protein-protein and protein-lipid interactions in this system. Fluorescence energy transfer (FRET) using MBP-II(Glc)-YFP and MBP-II(Glc)-CFP revealed that the homodimeric Enzyme II complex in cell membranes is stable (FRET(-)) but can be dissociated and reassociated to the heterodimer only in the presence of Triton X100 (FRET(+)). The monomeric species could form a heterodimeric species (FRET(+)) by incubation and purification without detergent exposure. Formaldehyde cross linking studies, conducted both in vivo and in vitro, revealed that the dimeric MBP-II(Glc) activity decreased dramatically with increasing formaldehyde concentrations due to both aggregation and activity loss, but that the monomeric MBP-II(Glc) retained activity more effectively in response to the same formaldehyde treatments, and little or no aggregation was observed. Electron microscopy of MBP-II(Glc) indicated that the dimeric form is larger than the monomeric form. Dynamic light scattering confirmed this conclusion and provided quantitation. NMR analyses provided strong evidence that the dimeric form is present primarily in a lipid bilayer while the monomeric form is present as micelles. Finally, lipid analyses of the different fractions revealed that the three lipid species (PE, PG and CL) are present in all fractions, but the monomeric micellar structure contains a higher percentage of anionic lipids (PG & CL) while the dimeric bilayer form has a higher percentage of zwitterion lipids (PE). Additionally, evidence for a minor dimeric micellar species, possibly an intermediate between the monomeric micellar and the dimeric bilayer forms, is presented. These results provide convincing evidence for interconvertible physical forms of Enzyme-II(Glc). |
| Links |
PubMed PMC3174158 Online version:10.1371/journal.pone.0024088 |
| Keywords |
Biological Transport; Biophysics/methods; Cell Membrane/metabolism; Cross-Linking Reagents/pharmacology; Cryoelectron Microscopy/methods; Cytoplasm/metabolism; Dimerization; Escherichia coli/enzymology; Fluorescence Resonance Energy Transfer/methods; Glucose/chemistry; Lipids/chemistry; Magnetic Resonance Spectroscopy/methods; Micelles; Octoxynol/pharmacology; Phosphotransferases/chemistry |
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