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.
Minarik
Antonín
A
Tomas Bata University in Zlín, Institute of Physics and Material Engineering, Mostní 5139, 760 01 Zlín, Czech Republic.
Humenik
Martin
M
Li
Sheng
S
Huang
Yiwei
Y
Krausch
Georg
G
Sprinzl
Mathias
M
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