Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Aluminum Silicates / chemistry*
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Bacterial Proteins
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Binding Sites
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Esterases / antagonists & inhibitors
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Esterases / chemistry*
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Esterases / genetics
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Esterases / ultrastructure*
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Gene Expression
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Humans
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Ketones / chemistry
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Ketones / pharmacology
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Ligands
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Microscopy, Atomic Force*
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Multienzyme Complexes / chemistry
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Multienzyme Complexes / genetics
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NADH, NADPH Oxidoreductases / chemistry
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NADH, NADPH Oxidoreductases / genetics
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Nucleocytoplasmic Transport Proteins / chemistry
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Nucleocytoplasmic Transport Proteins / genetics
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Protein Structure, Tertiary
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / ultrastructure*
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Thermus thermophilus / enzymology
Substances
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Aluminum Silicates
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Bacterial Proteins
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Ketones
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Ligands
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Multienzyme Complexes
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Nucleocytoplasmic Transport Proteins
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Recombinant Fusion Proteins
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NADH oxidase
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NADH, NADPH Oxidoreductases
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Esterases
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mica