Ligand-directed immobilization of proteins through an esterase 2 fusion tag studied by atomic force microscopy
Language English Country Germany Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- Bacterial Proteins MeSH
- Esterases antagonists & inhibitors chemistry genetics ultrastructure MeSH
- Gene Expression MeSH
- Ketones chemistry pharmacology MeSH
- Humans MeSH
- Ligands MeSH
- Microscopy, Atomic Force * MeSH
- Multienzyme Complexes chemistry genetics MeSH
- NADH, NADPH Oxidoreductases chemistry genetics MeSH
- Nucleocytoplasmic Transport Proteins chemistry genetics MeSH
- Recombinant Fusion Proteins chemistry genetics ultrastructure MeSH
- Aluminum Silicates chemistry MeSH
- Protein Structure, Tertiary MeSH
- Thermus thermophilus enzymology MeSH
- Binding Sites MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Bacterial Proteins MeSH
- Esterases MeSH
- Ketones MeSH
- Ligands MeSH
- mica MeSH Browser
- Multienzyme Complexes MeSH
- NADH oxidase MeSH Browser
- NADH, NADPH Oxidoreductases MeSH
- Nucleocytoplasmic Transport Proteins MeSH
- Recombinant Fusion Proteins MeSH
- Aluminum Silicates MeSH
Atomically flat mica surfaces were chemically modified with an alkyl trifluoromethyl ketone, a covalent inhibitor of esterase 2 from Alicyclobacillus acidocaldarius, which served as a tag for ligand-directed immobilization of esterase-linked proteins. Purified NADH oxidase from Thermus thermophilus and human exportin-t from cell lysates were anchored on the modified surfaces. The immobilization effectiveness of the proteins was studied by atomic force microscopy (AFM). It was shown that ligand-esterase interaction allowed specific attachment of exportin-t and resulted in high-resolution images and coverage patterns that were comparable with immobilized purified protein. Moreover, the biological functionality of immobilized human exportin-t in forming a quaternary complex with tRNA and the GTPase Ran-GTP, and the dimension changes before and after complex formation were also determined by AFM.
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