Subunit structure of functional porin oligomers that form permeability channels in the other membrane of Escherichia coli.

T Nakae,J Ishii,M Tokunaga

doi:10.1016/s0021-9258(17)37790-6

Abstract

Oligomers of a protein, porin, form permeability channels in the outer membrane of Escherichia coli B. A functional porin oligomer was identified and was purified to homogeneity by gel filtration in the presence of salts and sodium dodecyl sulfate. Molecular weights of purified porin oligomer and heat-dissociated monomer appeared to be 102,900 and 32,600, respectively, when determined by sedimentation equilibrium in the presence of sodium dodecyl sulfate. We concluded that the porin oligomer thus consists of three identical subunits. These data and results from other laboratories suggest porin trimers exist also in the outer membrane of intact cells, and participate in the formation of permeability channels. It was found that porin trimer bound less sodium dodecyl sulfate than the porin monomer.

Highlights

Subunit Structure of Functional Porin Oligomers That Form Permeability Channels in the Outer Membrane of Escherichia coZi*
It was found that porin trimer bound less sodium dodecyl sulfate than the porin monomer
Since the column eluates contained small oligomers of porin, we asked whether this porin oligomer still retained the ability to confer permeability channels when included in the reconstituted vesicles

Summary

AND METHODS

Bacterial Strain-The bacterial strain used in this study was E. The peptidoglycan-associated proteins (about 35 mg), after reducing SDS by repeated centrifugations, were suspended in 1.8 ml of the solution containing 0.1 M sodium phosphate buffer, pH 7.4,360 pg of lysozyme, and 3 mM NaN3, and was incubated at 37°C for 2 h as described previously [7]. Determination of Detergent Binding to Proteins-For equilibrium dialysis, Visking bags each containing 0.01 to 0.75% [““SISDS (specific activity of 100 pCi/2.5 g of SDS), 0.25 mg to 1.5 mg of purified porin, and 3 rnM NaNs in a total volume of 0.5 ml were dialyzed for 3 weeks against 20 ml of appropriate solutions containing [““SISDS of the same specific activity as described in the legend to Fig. 5.

Permeability Channels in Outer Membrane

RESULTS AND DISCUSSION

MOLECULAR WEIGHT

TABLE I

Oligomers treated with

LOG CONCENTRATION