Probing intra-molecular mechanics of carbonic anhydrase B with an atomic force microscope

Tong Wang, Hideo Arakawa and Atsushi Ikai

Laboratory of Biodynamics, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Japan 226-8501

We used an atomic force microscope (AFM) to investigate the intra-molecular mechanics of bovine carbonic anhydrase B (CAB) at the single molecular level. The protein was covalently sandwiched between a silanized silicon substrate and an AFM tip using bifunctional cross-linkers. The force required to unfold the protein was then measured under various conditions. A monomeric CAB could not be unfolded beyond ca. 15 nm at a maximum available tensile force of 1.7 nN, probably due to the presence of a 'knot' structure close to the C-terminus. When a tandemly repeated dimeric CAB was stretched under the same condition, a presumably knotless half of the molecule was extended to its maximal length supporting our conjecture. Two transient forms were detected during unfolding/refolding cycles for which strong segmental interaction forces, though different in nature, were observed. When the effect of a specific inhibitor on the force curve was examined, we found to our surprise that the knotless half of the dimer CAB had a capacity to interact with the inhibitor but was enzymatically not active.