Biochemical and biophysical characterization of the cell wall porin of Corynebacterium glutamicum: the channel is formed by a low molecular mass polypeptide.
The cell wall of the Gram-positive bacterium Corynebacterium glutamicum contains a channel (porin) for the passage of hydrophilic solutes. The channel-forming protein was identified, by lipid bilayer experiments, in the cell envelope fractions isolated by sucrose-density centrifugations and in organic solvent of whole cells. It was purified to homogeneity by fast-protein liquid chromatography across a Mono-Q column. The pure protein had a rather low molecular mass of about 5 kDa as judged by SDS-PAGE, which suggested that the cell wall channel is formed by a protein oligomer. The monomer has according to partial sequencing no significant homology to known protein sequences. The purified protein formed large ion-permeable channels in lipid bilayer membranes from phosphatidylcholine/phosphatidylserine mixtures with a single-channel conductance of 5.5 nS in 1 M KCl. Experiments with different salts suggested that the cell wall channel of C. glutamicum was highly cation-selective caused by negative charges localized at the channel mouth. The analysis of the single-channel conductance data using the Renkin correction factor suggested that the diameter of the cell wall channel is about 2.2 nm. Channel-forming properties of the cell wall channel of C. glutamicum were compared with those of mycobacteria. These channels share common features because they form large and water-filled channels that contain point net charges.
Lichtinger
T
T
Lehrstuhl für Biotechnologie, Biozentrum der Universität Würzburg, Germany.
Burkovski
A
A
Niederweis
M
M
Krämer
R
R
Benz
R
R
D000595:Amino Acid Sequence; D002463:Cell Membrane Permeability; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D003352:Corynebacterium / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D019286:Endopeptidase K / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology; D008051:Lipid Bilayers / Q000378:metabolism; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology; D018272:Porins / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology
D007473:Ion Channels; D008051:Lipid Bilayers; D010446:Peptide Fragments; D010455:Peptides; D018272:Porins; D019286:Endopeptidase K