1. Properties of chemically modified porin from Escherichia coli in lipid bilayer membranes
R Benz, H Tokunaga, T Nakae Biochim Biophys Acta. 1984 Jan 25;769(2):348-56. doi: 10.1016/0005-2736(84)90316-x.
Purified porin OmpF from Escherichia coli outer membrane was chemically modified by acetylation and succinylation of amino groups and by amidation of the carboxyl groups. Native and chemically modified porins were incorporated into lipid bilayer membranes and the permeability properties of the pores were studied. Acetylation and succinylation of the porin trimers had almost no influence on the single channel conductance in the presence of small cations and anions and the cation selectivity remained essentially unchanged as compared with the native porin. Amidation had also only little influence on the single channel conductance and changed the pore conductance at maximum by less than 50%, whereas the cation selectivity of the porin is completely lost after amidation. The results suggest that the structure of the porin pore remains essentially unchanged after chemical modification of the pores and that their cation selectivity is caused by an excess of negatively charged groups inside the pore and/or on the surface of the protein. Furthermore, it seems very unlikely that the pore contains any positively charged group at neutral pH.
2. The mechanism of ion selectivity of OmpF-porin pores of Escherichia coli
Y Kobayashi, T Nakae Eur J Biochem. 1985 Sep 2;151(2):231-6. doi: 10.1111/j.1432-1033.1985.tb09093.x.
The OmpF porin from the outer membrane of Escherichia coli acts as a lightly cation-selective pore, allowing the diffusion of small cations and cationic molecules, whose Mr are a little larger than the threshold exclusion limit. To ascertain the mechanism of this cation selectivity, we have examined a possible influence of cationic solutes on the fluorescence emission and the circular dichroic spectrum of tryptophan residues of the porin trimer, searching for conformational change(s). The diffusion of cationic solutes was determined with the native and the amidated porins in the presence or the absence of the effector cations. The following results were obtained. (a) Cations, e.g. spermidine, caused fluorescence quenching in the native trimer, with a half-maximum fluorescence quenching at 11-18 microM. A change in the circular dichroic spectrum was also recorded at around 280 nm. (b) The dissociation constant of spermidine to the native trimer was calculated to be 16 microM as determined by the method of equilibrium dialysis. (c) The cation-caused fluorescence quenching was reversed when the carboxyl groups of the trimer were modified by the amidation reaction, though amidation of the trimer resulted in no significant change in the fluorescence intensity. (d) The diffusion rate of N-benzyloxycarbonyl-glycyl-L-prolyl-L-arginine p-nitroanilide through the native and the amidated porins was lowered in the presence and the absence, respectively, of cations. Both the extent of fluorescence quenching in the presence of cation and the rate of cation diffusion were inversely proportional to the number of amidated carboxyl residues. The relative fluorescence quenching of the porin trimer (the amidated versus the native) in the presence of cations was linearly related to the relative solute diffusion via the porin (the amidated versus the native). These results suggested that cations caused a conformational change in the trimer, resulting in an easier diffusion of the solutes. The results suggested further that a limited number of carboxyl groups in the pore interior are involved in the cation selectivity of OmpF-porin pores.
3. The solute selectivity of porin pores of Escherichia coli and Salmonella typhimurium
T Nakae, J N Ishii, H Tokunaga, Y Kobayashi, R Nakae Tokai J Exp Clin Med. 1982;7 Suppl:141-8.
The solute selectivity of porin pores of Escherichia coli and Salmonella typhimurium was examined using vesicle membranes reconstituted from phospholipids and purified porin trimers. OmpE- and 34K-porin-pores allowed a preferential diffusion of phosphorylated and/or negatively charged compounds. They allowed the diffusion of positively charged solutes reasonably well. OmpF- and 35K-porin-pores favored the diffusion of positively charged solutes a little more than negatively charged solutes. OmpC- and 36K-porin-pores showed poor diffusibility to all solute tested. B-porins from E. coli B formed highly efficient pores for the diffusion of both positively and negatively charged solutes. On the basis of these observations, possible mechanisms of solute selectivity were discussed.
