Identification of the outer membrane porin of Thermus thermophilus HB8: the channel-forming complex has an unusually high molecular mass and an extremely large single-channel conductance

Abstract

The outer membrane of the thermophilic bacterium Thermus thermophilus was isolated using sucrose step gradient centrifugation. Its detergent extracts contained an ion-permeable channel with an extremely high single-channel conductance of 20 nS in 1 M KCl. The channel protein was purified by preparative sodium dodecyl sulfate (SDS)-polyacylamide gel electrophoresis. It has a high molecular mass of 185 kDa, and its channel-forming ability resists boiling in SDS for 10 min.

FIG. 1

SDS–7% PAGE of the supernatant of cell envelopes treated with 10 mM Tris-HCl, 10 mM CaCl₂ (pH 8), and 0.6% LDAO. Lane 1, molecular mass markers (66, 45, and 36 kDa). Lane 2, 50 μg of protein of the supernatant was solubilized at 30°C for 10 min in 15 μl of sample buffer. The gel was stained with Coomassie brilliant blue.

FIG. 2

SDS–5% PAGE of the 185-kDa protein of the outer membrane of T. thermophilus obtained by elution from preparative SDS-polyacrylamide gels. Lane 1, high-molecular-mass markers (205, 116, 97, 84, and 66 kDa). Lane 2, 5 μg of the pure protein solubilized at 100°C for 10 min in 7 μl of sample buffer. The gel was stained with Coomassie brilliant blue.

FIG. 3

Single-channel recording of the 185-kDa porin from the outer membrane of T. thermophilus. Channel-forming activity was measured with lipid bilayer membranes made of a 1% (wt/vol) solution of diphytanoyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala.) in n-decane (2). The aqueous phase contained 1 M KCl and 5 ng of protein per ml. The applied membrane potential was 20 mV; T = 20°C. The single-channel recordings were performed using Ag/AgCl electrodes (with salt bridges) connected in series to a voltage source and a Keithley 427 current amplifier. The amplified signal was monitored on a digital oscilloscope and recorded on a strip chart or tape recorder.