PMID:9425040

Citation	van der Does, C, Manting, EH, Kaufmann, A, Lutz, M and Driessen, AJ (1998) Interaction between SecA and SecYEG in micellar solution and formation of the membrane-inserted state. Biochemistry 37:201-10
Abstract	Preprotein translocation in Escherichia coli is mediated by the translocase with SecA as peripheral ATPase and SecY, SecE, and SecG as membrane domain. To facilitate large-scale purification of the SecYEG heterotrimer, SecY was fused at its amino terminus with a hexahistidine tag and co-overexpressed with SecE and SecG. The presence of the His tag allowed purification of homogeneously pure SecYEG complex by a single anion-exchange chromatographic step starting from octyl glucoside-solubilized inner membranes. Endogenous levels of SecD and SecF copurified with the SecYEG protein. Purified SecYEG complex retained a nativelike, alpha-helical conformation in octyl glucoside and in micellar solution binds SecA with high affinity. In the presence of the nonhydrolyzable nucleotide analogue adenosine 5'-(beta, gamma-imidotriphosphate), octyl glucoside-solubilized SecYEG is nearly as effective as the reconstituted enzyme in inducing the formation of a proteinase K-protected 30 kDa fragment of 125I-labeled SecA, while SecYEG is proteolyzed to fragments smaller than 6 kDa. These data demonstrate that the 30-kDa SecA fragment is not protected by the lipid phase nor by SecYEG but rather indicate that it represents a SecYEG- and nucleotide-induced stable conformational state of a SecA domain.
Links	PubMed Online version:10.1021/bi972105t
Keywords	Adenosine Triphosphatases/chemistry; Adenosine Triphosphatases/metabolism; Adenosine Triphosphate/physiology; Amino Acid Sequence; Bacterial Proteins/chemistry; Bacterial Proteins/isolation & purification; Bacterial Proteins/metabolism; Biological Transport; Detergents; Enzyme Stability; Escherichia coli/enzymology; Escherichia coli Proteins; Glucosides; Macromolecular Substances; Membrane Proteins/chemistry; Membrane Proteins/isolation & purification; Membrane Proteins/metabolism; Membrane Transport Proteins; Micelles; Molecular Sequence Data; Operon; Protein Binding; Protein Structure, Secondary; Solubility
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