PhoE porin

Typically, functional porins are homotrimers, which assemble from monomers and then integrate into the outer membrane. The general porins, water-filled diffusion pores, allow the passage of hydrophilic molecules up to a size of approximately 600 Daltons. They do not show particular substrate specificity, but display some selectivity for either anions or cations, and some discrimination with respect to the size of the solutes. The first published crystal structure of a bacterial porin was that of R. capsulatus [48]. Together with the atomic structures of two proteins from E. coli, the phosphate limitation-induced anion-selective PhoE porin and the osmotically regulated cation-selective OmpF porin, a common scheme was found [49]. Each monomer consists of 16 (3-strands spanning the outer membrane and forming a barrel-like structure. 

Figure 8-20 MolScript ribbon drawings of the OmpF porin of E. coli. (A) View of the 340-residue monomer. (B) View of the trimer looking down the threefold axis. From Wa-tanabe et al3is From atomic coordinates of Cowan et al.3i9 (C) Molecular model of the constriction zone of the PhoE porin. Locations of key residues are shown, with positions of homologous residues in OmpF given in parentheses. Extracellular loops have been omitted. Constructed from coordinates of Cowan et al.349 by Samartzidou and Del-cour.342 Courtesy of Anne Delcour.

Phosphate limitation of E. coli K12 cultures results in a derepression of a protein, PhoE, whose apparent function is to scavenge trace amounts of phosphorous-containing nutrients from this environment. The PhoE protein is immunologically related to OmpC and OmpF and seems to be produced at their expense under phosphate-limitation. The PhoE porin exhibits a preference for anionic solutes, particularly pyrophosphates. The protein bears a specific phosphate binding site. The PhoE protein facilitates the growth of the organism on polyphosphate, provided as the sole source of phosphorus. 

SELECTIVE DETERMINATION OF PHOSPHATE WITH PhoE PORIN-LECITHIN MEMBRANE ELECTRODE... 

The electrode system for the characterization of the PhoE porin-lecithin membrane is depicted in Figure 1. A basal plane pyrolytic graphite (BPG) electrode was coated with a PhoE porin-lecithin membrane. The electrode system consisted of the PhoE porin-lecithin membrane BPG electrode with a surface area of 0.19 cm, a counter electrode (platinum wire), and a reference electrode (saturated calomel electrode S.C.E.). Cyclic voltammograms were obtained with a potentiostat (Hokuto Denko,... 

Model HA301), a function generator (Hokuto Denko, Model HB104) and an X-Y recorder (Riken Denshi, F35). The measurement cell was of all-glass construction, approximately 10 ml in volume, incorporating a conventional three-electrode system. An anion-selective polymer membrane electrode was also coated with a PhoE porin-lecithin membrane. Potentiometric measurements were made with an electrometer (Hokuto Denko, Model HE-IOIA) in conjunction with a recorder (Riken Denshi, Model SP-J3C). 

Figure 1. Schematic diagram of the PhoE porin-lecithin membrane-BPG electrode system for measurement of phosphocompounds.

The resultant PhoE porin was checked by SDS-polyacrylamide gel electrophoresis. The amount of PhoE porin was determined with the Bio-Rad Protein Assay Bio-Rad, Richmond, CA). The extracted PhoE porin solution was dialyzed against 4L of 50 mM Tris-HCl buffer (pH 7.2) containing 3 mM NaNs before using. 

A PhoE porin-lecithin membrane-BPG electrode was prepared as follows n-decane containing 0.5% egg lecithin and 0.25% cholesterol was brushed on the BPG electrode and dried in air. The resulting lecithin membrane-BPG electrode was inserted in 10 ml, 50 mM Tris-HCL buffer (pH 7.0), and coated again with the n-decane solution containing lecithin and cholesterol. After the lecithin membrane turned black, extracted PhoE porin was added to the lecithin membrane-BPG electrode and Tris-HCl buffer solution system. The anion-selective polymer membrane electrode was an Ag/AgCl electrode (0.422 cm2) coated with a PVC membrane containing 6% methyltridodecyl ammonium chloride and 30% nitrophenyloctyl ether. A PhoE porin-lecithin membrane-anion selective membrane electrode was prepared in the same way as the PhoE porin-lecithin membrane-BPG electrode described above. 

The PhoE porin-lecithin membrane electrodes were inserted in the sample solution. Cyclic voltammetry was run in the range -1.0 to 1.0 V vs SCE. The potential was measured in combination with the saturated calomel electrode with an electrometer and displayed on a recorder. 

Selectivity of PhoE porin-lecithin membrane-BPG electrode... 

The selectivity of the PhoE porin-lecithin membrane electrode is indicated in Table 1. The cyclic voltammograms were obtained at the PhoE membrane electrode for L-Cysteine, riboflavin, FMN, NADH and FADH2. Peak currents were obtained for FMN. L-cysteine and riboflavin, which have no phosphate, did not show a peak current. Peak currents were not obtained for NADH and FADH2. The molecular weights of NADH and FADH2 were more than 700. The PhoE porin-lecithin membrane is apparently permeable to phospho-compounds having molecular weights less than 700 daltons. 

Effect of amount of PhoE porin on peak current... 

Figure 4 shows the relationship between the peak current of FMN and the amount of PhoE porin in the lecithin membrane-BPG electrode. The peak... 

Figure 3. Cyclic voltammograms of riboflavin at a PhoE porin-lecithin membrane-BPG electrode and lecithin membrane electrode. Both voltammograms are similar. Scan rate was 10 mV/s. Riboflavin concentration was 60 pM. The experiments were performed in 50 mM Tris-HCl buffer (pH 7.0).

Table 1. Redox potentials of cyclic voltammograms of electroactive substrates observed at a PhoE porin-lecithin membrane BPG electrode. 

Phosphate was measured with the anion-selective electrode coated with a PhoE porin-lecithin membrane. When phosphate solution was added to the... 

Figure 4. Relationship between peak current and PhoE porin concentration in buffer inserted PhoE porin-lecithin membrane-BPG electrode. FMN concentration was 0.44 mM and experiments were performed in 0.1 M Tricine buffer (pH 7.4), 25 C.

PhoE-lecithin membrane electrode system the potential changed until it reached a maximum value. The maximum value was attained in all cases within 10 min, and the maximum potential was dependent on the phosphate concentration. When the PhoE porin-lecithin electrode system was removed from the sample solution and placed in Tricine buffer solution, the potential returned to its initial level. 

The selectivity of the PhoE porin-lecithin membrane electrode was studied for acetate, nitrate, nitrite, formate, citrate, succinate, propionate, chloride and phosphate. The potential changes were 6.5 mV for phosphate and 1.2 mV for chloride when the concentrations of ions were 3.5 mM. No potential change was obtained for acetate, nitrate, nitrite, formate, succinate, and propionate. Therefore, phosphate can be selectively determined in the absence of chloride. 

Figure 5 shows the relationship between the potential change in the PhoE porin-lecithin membrane electrode and the concentration of phosphate. The potential increased with increasing phosphate concentration. A linear relationship was obtained between the potential change and the logarithm of phosphate concentration in the range 0.2 - 9 mM. 

Selective Determination of Phosphate with PhoE Porin-Lecithin Membrane Electrode... 

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