Ligand-directed immobilization of proteins through an esterase 2 fusion tag studied by atomic force microscopy.
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.
Tomas Bata University in Zlín, Institute of Physics and Material Engineering, Mostní 5139, 760 01 Zlín, Czech Republic.
Antonín
A
Minarik
Martin
M
Humenik
Sheng
S
Li
Yiwei
Y
Huang
Georg
G
Krausch
Mathias
M
Sprinzl
D000538:Aluminum Silicates / Q000737:chemistry*; D001426:Bacterial Proteins; D001665:Binding Sites; D004950:Esterases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*; D015870:Gene Expression; D006801:Humans; D007659:Ketones / Q000737:chemistry / Q000494:pharmacology; D008024:Ligands; D018625:Microscopy, Atomic Force; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics; D029543:Nucleocytoplasmic Transport Proteins / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure*; D016963:Thermus thermophilus / Q000201:enzymology
D000538:Aluminum Silicates; D001426:Bacterial Proteins; D007659:Ketones; D008024:Ligands; D009097:Multienzyme Complexes; D029543:Nucleocytoplasmic Transport Proteins; D011993:Recombinant Fusion Proteins; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D004950:Esterases; C011934:mica