PMID:10196117
| Citation |
Zhuang, J, Privé, GG, Werner, GE, Ringler, P, Kaback, HR and Engel, A (1999) Two-dimensional crystallization of Escherichia coli lactose permease. J. Struct. Biol. 125:63-75 |
|---|---|
| Abstract |
A chimeric protein consisting of lactose permease with cytochrome b562 in the middle cytoplasmic loop and six His residues at the C terminus (LacY/L6cytb562/417H6 or "red permease") was overexpressed in Escherichia coli and isolated by nickel affinity chromatography after solubilization with dodecyl-beta,d-maltopyranoside. Red permease was then reconstituted in the presence of phospholipids, yielding densely packed vesicles and well-ordered two-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 reconstitution conditions, trigonal and rectangular crystallographic packing arrangements 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 maps calculated to a resolution of 2 nm show that these crystals consist of two layers. |
| Links |
PubMed Online version:10.1006/jsbi.1998.4059 |
| Keywords |
Carrier Proteins/ultrastructure; Crystallization; Cytochrome b Group/ultrastructure; Escherichia coli/enzymology; Escherichia coli Proteins; Lipids/analysis; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/ultrastructure; Microscopy, Electron; Models, Molecular; Monosaccharide Transport Proteins; Phospholipids/metabolism; Recombinant Fusion Proteins/ultrastructure; Symporters |
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