期刊
JOURNAL OF STRUCTURAL BIOLOGY
卷 152, 期 2, 页码 129-139出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jsb.2005.07.010
关键词
Perfringolysin O; cryo-electron microscopy; nickel-lipid nanotubes; helical arrays; 3D density maps
资金
- NIAID NIH HHS [AI037657, R01 AI037657] Funding Source: Medline
- NIAMS NIH HHS [AR39155] Funding Source: Medline
- NIGMS NIH HHS [R01 GM061938, GM61938, GM52468] Funding Source: Medline
To facilitate purification and subsequent structural studies of recombinant proteins the most widely used genetically encoded tag is the histidine tag (His-tag) which specifically binds to N-nitrilotriacetic-acid-chelated nickel ions. Lipids derivatized with a nickel-chelating head group can be mixed with galactosylceramide glycolipids to prepare lipid nanotubes that bind His-tagged proteins. In this study, we use His-tagged perfringolysin O (PFO), a soluble toxin secreted by the bacterial pathogen Clostridium perfringens, as a model protein to test the utility of nickel-lipid nanotubes as a tool for structural studies of His-tagged proteins. PFO is a member of the cholesterol dependent cytolysin family (CDC) of oligomerizing, pore-forming toxins found in a variety of Gram-positive bacterial pathogens. CDC pores have been difficult to study by X-ray crystallography because they are membrane associated and vary in size. We demonstrate that both a wild-type and a mutant form of PFO form helical arrays on nickel-lipid containing nanotubes. Cryo-electron microscopy and image analysis of the helical arrays were used to reconstruct a 3D density map of wild-type PFO. This study suggests that the use of nickel-lipid nanotubes may offer a general approach for structural studies of recombinant proteins and may provide insights into the molecular interactions of proteins that have a natural affinity for a membrane surface. (C) 2005 Elsevier Inc. All rights reserved.
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