THE PORIN RAFY ENCODED BY THE RAFFINOSE PLASMID PRSD2 OF ESCHERICHIA-COLI FORMS A GENERAL DIFFUSION PORE AND NOT A CARBOHYDRATE-SPECIFIC PORIN

The gene rafY from the plasmid pRSD2, which enables Escherichia coli t o grow on raffinose, was transferred into expression plasmid pUSL77. T he protein was expressed in the porin-deficient Escherichia coli strai n KS26 and was isolated and purified to homogeneity. The pure protein was reconstituted into lipid bilayer membranes. It formed an ion-perme able channel with a single-channel conductance of 2.9 nS of the open s tate in 1 M KCl, which is approximately twice of that of the general d iffusion pores OmpF and OmpC of E. coli outer membrane. At lower pH th e channel exhibited rapid flickering between three substates of the op en channel. The RafY channel appears to be wide and water filled and h as a small selectivity for cations over anions. Although RafY is part of an uptake and fermentation system for raffinose it does not contain a binding site for carbohydrates. Our results suggest that RafY is a general diffusion pore with a diameter, larger than that of the genera l diffusion porins OmpF and OmpC, that allows the diffusion of high-mo lecular-mass carbohydrates through the outer membrane.