Three Dimensional Structure of a Membrane Pore: Electron Microscopical Analysis of Escherichia coli Outer Membrane Matrix Porin

Abstract

Porin (the product of gene ompF) is an integral protein (MW 36,500) spanning the outer membrane of Escherichia coli and may be regarded as a paradigm for pores which allow diffusion of solutes smaller than 650d into cells (1). We have investigated its three-dimensional structure by electron microscopy to characterize its pore geometry in an attempt to relate its structure to its function. The protein has also been characterized biochemically (2) and functionally (3, 4). All structural studies (5) indicate trimers as the basic unit, consistent with functional behavior, inasmuch as pore channels are activated as multiples of three unit steps (3). For the experiments presented here we have utilized vesicles which were reconstituted in the presence of dimyristoyl phosphatidylcholine after removal of octyl-POE (octyl-oligooxyethylene), which had been used for solubilization and purification of porin (5). Electron-microscopic and diffraction measurements were made on uranyl acetate negatively stained specimens and, more recently, on sugar-embedded preparations (resolution 20 A and 13.7 A,). All phase information, extracted from images obtained in tilt series (± 600) was reduced to a common origin via cross-correlation in direct space using the SEMPER image analysis system (6). Combination with electron diffraction intensity data allowed us to obtain three-dimensional images. Recent theoretical calculations have justified this approach by showing that n-beam dynamic pertubations are not important for diffraction intensities from thin negatively-stained membrane preparations (7).