Two-dimensional crystallization of Escherichia coli lactose permease

A chimeric protein consisting of lactose permease with cytochrome b(562) in the middle cytoplasmic loop and six His residues at the C terminus (LacY/L 6cytb(562)/417H6 or "red permease") was overexpressed in Escherichia coli a nd isolated by nickel affinity chromatography after solubilization with dod ecyl-beta,D-maltopyranoside. Red permease was then reconstituted in the pre sence of phospholipids, yielding densely packed vesicles and well-ordered t wo dimensional (2D) crystals as shown by electron microscopy of negatively stained specimens. Single-particle analysis of 16 383 protein particles in densely packed vesicles reveals a 5,4-nm-long trapeziform protein of 4.1 to 5.1 nm width, with a central stain-filled indentation. Depending on recons titution conditions, trigonal and rectangular crystallographic packing arra ngements of these elongated particles assembled into trimers are observed, The best ordered 2D crystals exhibit a rectangular unit cell, of dimensions a = 9.9 nm, b = 17.4 nm, that houses two trimeric complexes. Projection ma ps calculated to a resolution of 2 nm show that these crystals consist of t wo layers. (C) 1999 Academic Press.