title: Force measurement and inhibitor binding assay of monomer and engineered dimer of bovine carbonic anhydrase B.
abstract: We applied atomic force microscopy (AFM) to study the intramolecular mechanics of the globular protein molecule, bovine carbonic anhydrase B. The immobilized protein on an amino-functionalized silicon wafer was pulled from its N- and C-termini after being covalently cross-linked to the AFM tip, and the relationship between the tensile force applied on the protein and its extension was recorded. The native enzyme (having 261 residues with two Cys added at its ends, and in a theoretical stretching length of 96 nm) was extended only to 13 +/- 2 nm under physiological conditions before disruption of the covalent cross-linking system. Contrary to the above observation, an engineered dimer was extended to about 110 nm even in the absence of the denaturant. The difference was ascribed to the presence or presumed absence of a "knot" structure at the C-terminal end of the two forms, respectively. When a specific inhibitor was added to the experimental solution, native monomers (sp activity = 88% of the wild type enzyme) were extended to 28 +/- 4 nm, whereas dimers (sp activity = 46%) were extended to about 56 +/- 3 nm, suggesting that both monomeric units in the dimer could bind inhibitor molecules, which was further corroborated by a titration experiment using a fluorescent inhibitor. Thus, one of the monomeric units in the engineered dimer was concluded to be enzymatically inactive but capable of binding inhibitors.
authors: Laboratory of Biodynamics, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan.
authors: T
authors: T
authors: Wang
authors: H
authors: H
authors: Arakawa
authors: A
authors: A
authors: Ikai
mesh_terms: D000818:Animals; D002257:Carbonic Anhydrase Inhibitors / Q000737:chemistry*; D002256:Carbonic Anhydrases / Q000737:chemistry*; D002417:Cattle; D018360:Crystallography, X-Ray; D019281:Dimerization; D018625:Microscopy, Atomic Force
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D002256:Carbonic Anhydrases