Biochemical identification and biophysical characterization of a channel-forming protein from Rhodococcus erythropolis.
Organic solvent extracts of whole cells of the gram-positive bacterium Rhodococcus erythropolis contain a channel-forming protein. It was identified by lipid bilayer experiments and purified to homogeneity by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The pure protein had a rather low molecular mass of about 8.4 kDa, as judged by SDS-PAGE. SDS-resistant oligomers with a molecular mass of 67 kDa were also observed, suggesting that the channel is formed by a protein oligomer. The monomer was subjected to partial protein sequencing, and 45 amino acids were resolved. According to the partial sequence, the sequence has no significant homology to known protein sequences. To check whether the channel was indeed localized in the cell wall, the cell wall fraction was separated from the cytoplasmic membrane by sucrose step gradient centrifugation. The highest channel-forming activity was found in the cell wall fraction. The purified protein formed large ion-permeable channels in lipid bilayer membranes with a single-channel conductance of 6.0 nS in 1 M KCl. Zero-current membrane potential measurements with different salts suggested that the channel of R. erythropolis was highly cation selective because of negative charges localized 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 channel-forming properties of the cell wall channel of R. erythropolis were compared with those of other members of the mycolata. These channels have common features because they form large, water-filled channels that contain net point charges.
Lichtinger
T
T
Lehrstuhl für Biotechnologie, Biozentrum der Universität Würzburg, D-97074 Würzburg, Germany.
Reiss
G
G
Benz
R
R
D002463:Cell Membrane Permeability; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D007473:Ion Channels / Q000502:physiology*; D008564:Membrane Potentials; D008970:Molecular Weight; D012240:Rhodococcus / Q000502:physiology*
D007473:Ion Channels