Channel-forming (Porin) activity in Herpetosiphon aurantiacus Hp a2

Abstract

Detergent extracts of cell envelopes of the gliding bacterium Herpetosiphon aurantiacus formed channels in lipid bilayers. Fast protein liquid chromatography across a HiTrap-Q cation-exchange column demonstrated that a 45-kDa protein forms the channel. The observation of a channel-forming protein suggests that Herpetosiphon aurantiacus Hp a2 has a permeability barrier on its surface.

FIG. 1.

SDS-PAGE (7% polyacrylamide), performed according to Laemmli (14), as part of the purification procedure for the 45-kDa channel-forming protein of H. aurantiacus Hp a2. The gel was stained with Coomassie brilliant blue, and all protein samples were treated for 10 min at 100°C. Lane 1, low-molecular-mass markers of 36, 45, and 66 kDa; lane 2, high-molecular-mass markers of 36, 45, 55, 66, 84, 97, 116, and 205 kDa; lane 3, 15 μl of the 0.4% LDAO extract of the cell envelope fraction dissolved in 10 μl of sample buffer; lane 4, 5 μg of protein of fraction 32 of the Hitrap-Q FPLC column dissolved in 15 μl of sample buffer.

FIG. 2.

Single-channel recording of a PC/n-decane membrane in the presence of pure 45-kDa protein of H. aurantiacus Hp a2. The aqueous phase contained 1 M KCl (pH 6) and 10-ng/ml protein. The applied membrane potential was 20 mV (temperature of 20°C).

FIG. 3.

Single-channel recording of a PC/n-decane membrane in the presence of pure 45-kDa protein of H. aurantiacus Hp a2. The aqueous phase contained 1 M NH₄Cl (pH 5) and 10-ng/ml protein. The applied membrane potential was 10 mV (temperature of 20°C). Note that the channels were transient under these conditions and open and closed in a single step, indicating that the channel is formed by a protein monomer.