Force measurement and inhibitor binding assay of monomer and engineered dimer of bovine carbonic anhydrase B

We applied atomic force microscopy (AFM) to study the intramolecular mechan ics of the globular protein molecule, bovine carbonic anhydrase B, The immo bilized protein on an amino-functionalized silicon wafer was pulled from it s 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 e xtension 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 ext ended 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 d enaturant, 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 m onomers (sp activity = 88% of the wild type enzyme) were extended to 28 +/- 4 nn, whereas dimers (sp activity = 46%) were extended to about 56 +/- 3 n m, suggesting that both monomeric units in the dimer could bind inhibitor m olecules, which was further corroborated by a titration experiment using a fluorescent inhibitor. Thus, one of the monomeric units in the engineered d imer was concluded to be enzymatically inactive but capable of binding inhi bitors. (C) 2001 Academic Press.