Stoichiometry and Substrate Affinity of the Mannitol Transporter, EnzymeIImtl, from Escherichia coli

Abstract

Uptake and consecutive phosphorylation of mannitol in Escherichia coli is catalyzed by the mannitol permease EnzymeIImtl. The substrate is bound at an extracellular-oriented binding site, translocated to an inward-facing site, from where it is phosphorylated, and subsequently released into the cell. Previous studies have shown the presence of both a high- and a low-affinity binding site with K(D)-values in the nano- and micromolar range, respectively. However, reported K(D)-values in literature are highly variable, which casts doubts about the reliability of the measurements and data analysis. Using an optimized binding measurement system, we investigated the discrepancies reported in literature, regarding both the variability in K(D)-values and the binding stoichiometry. By comparing the binding capacity obtained with flow dialysis with different methods to determine the protein concentration (UV-protein absorption, Bradford protein detection, and a LDH-linked protein assay to quantify the number of phosphorylation sites), we proved the existence of only one mannitol binding site per dimeric species of unphosphorylated EnzymeIImtl. Furthermore, the affinity of EnzymeIImtl for mannitol appeared to be dependent on the protein concentration and seemed to reflect the presence of an endogenous ligand. The dependency could be simulated assuming that >50% of the binding sites were occupied with a ligand that shows an affinity for EnzymeIImtl in the same range as mannitol.

Flavin Binding to the High Affinity Riboflavin Transporter RibU

“…The requirement that the substrate is co-purified with the transporter may be easily met for high affinity binding systems such as RibU, but proteins that bind their substrates with lower affinity may lose the substrate during the purification. Nonetheless, theoretical calculations have shown that substantial amounts of substrates may be co-purified with proteins regardless of their binding affinity, provided that the protein concentrations are kept high throughout the purification (10 -20-fold higher than the dissociation constant) (29). Large amounts of membrane proteins of high concentration are routinely purified for crystallization purposes, and therefore MALDI-TOF mass spectrometry may provide a general tool for substrate identification in structural genomics projects in which membrane proteins of unknown function are being crystallized.…”

Substrate-induced Conformational Changes in the Membrane-embedded IICmtl-domain of the Mannitol Permease from Escherichia coli, EnzymeIImtl, Probed by Tryptophan Phosphorescence Spectroscopy

“…The high resolution of the spectrum of Trp-97 without mannitol suggests that both Trp residues in the dimer of Trp-97 are in a similar microenvironment. The large difference in lifetime between free and mannitol-bound Trp-97 indicate that mannitol binding influences both Trp residues in the dimer of Trp-97 in a similar manner when mannitol interacts at the single binding site present in the dimer (20).…”

“…All single-Trp EII mtl mutants were purified using Ni-NTA affinity chromatography as described (20). To remove all traces of the tryptophan phosphorescence quencher histidine (used to elute the protein from Ni-NTA) from the EII mtl mutant preparations, pooled Ni-NTA fractions were diluted 5 times in buffer (25 mM Tris-HCl, pH 7.6, 2 mM reduced glutathione, plus 0.25% C 10 E 5 ), loaded onto Q-Sepharose, washed with 20 column volumes of the same buffer, and subsequently eluted in a single step using 4 column volumes of the above buffer, supplemented with 400 mM NaCl (Suprapur, Merck).…”

Localization of the Substrate-binding Site in the Homodimeric Mannitol Transporter, EIImtl, of Escherichia coli

“…Enzyme II proteins are known to be functional only as dimers. EII mtl forms very strong dimers when in the bilayer or when solubilized by detergent (7), and careful analysis established that dimeric wt EII mtl shows one high affinity binding site (K D ϭ ϳ100 nM) (17). The presence of a second site with a K D Ͻ 0.1 mM could be excluded.…”

“…5-FTrp was chosen rather than Trp because of its homogeneous fluorescent decay when incorporated into a protein (19,20). Because EII mtl is dimeric and harbors only one high affinity mannitol-binding site/dimer (17), azi-mannitol bound at the mannitol-binding site can in principle show FRET with one or both 5-FTrps in the dimer. Comparison of the time-resolved fluorescence decays of 5-FTrp labeled EII mtl in the presence of mannitol versus azi-mannitol informs whether azi-mannitol shows FRET with one or both 5-FTrp residues.…”