Biochemical identification and biophysical characterization of a channel-forming protein from Rhodococcus erythropolis

Organic solvent extracts of whole cells of the gram-positive bacterium Rhod ococcus erythropolis contain a channel-forming protein. It was identified b y lipid bilayer experiments and purified to homogeneity by preparative sodi um dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The pur e protein had a rather low molecular mass of about 8.4 kDa, as judged by SD S-PAGE. SDS-resistant oligomers with a molecular mass of 67 kDa were also o bserved, suggesting that the channel is formed by a protein oligomer. The m onomer was subjected to partial protein sequencing, and 45 amino acids were resolved. According to the partial sequence, the sequence has no significa nt homology to known protein sequences. To check whether the channel was in deed localized in the cell wall, the cell wall fraction was separated from the cytoplasmic membrane by sucrose step gradient centrifugation. The highe st channel-forming activity was found in the cell wall fraction. The purifi ed protein formed large ion-permeable channels in lipid bilayer membranes w ith a single-channel conductance of 6.0 nS in 1 M KCl. Zero-current membran e potential measurements with different salts suggested that the channel of R. erythropolis was highly cation selective because of negative charges lo calized at the channel mouth. The correction of single-channel conductance data for negatively charged point charges and the Renkin correction factor suggested that the diameter of the cell wall channel is about 2.0 nm. The c hannel-forming properties of the cell wall channel of R. erythropolis were compared with those of other members of the mycolata. These channels have c ommon features because they form large, water-filled channels that contain net point charges.