Reconstitution of a Porin from Mycobacterium smegmatis at HOPG covered with hydrophobic host layers

The unique stability and self-assembling properties of porin MspA, a channel protein isolated from Mycobacterium smegmatis, were established using three very different host systems: (1) A very simple and straightforward nanostructuring process was achieved by depositing buffer droplets containing MspA onto highly ordered pyrolytic graphite (HOPG) from the gas phase, followed by thermal curing and high vacuum treatment. Three lateral nanostructures were obtained depending on the protein mass deposited, the temperature during deposition and the subsequent curing process. We report here a detailed statistical analysis of the MspA pore sizes on HOPG. (2) The hydrophobic, water-soluble copolymer poly(N-isopropylacrylamide)-poly(acrylic acid) at HOPG was used as the host system on HOPG for the successful reconstitution of MspA from the liquid phase. The existence of hydrophilic nanopores, which formed exclusively when MspA was present, was detected at temperatures above the lower critical solution temperature of the copolymer. (3) The outer membrane of Mycobacterium tuberculosis at HOPG was employed as the hydrophobic host system for the channel porin from Mycobacterium smegmatis (MspA). Evidence for the reconstitution of MspA into the related membrane of M. tuberculosis was obtained. The nanostructures were analysed using high-resolution transmission electron microscopy. The program IMAGE (NIH, USA) permitted a quantitative insight into the lateral nanostructures at HOPG. Copyright (C) 2004 John Wiley Sons, Ltd.