Reversible stretching of a monomeric unit in a dimeric bovine carbonic anhydrase B with the atomic force microscope.

We have previously shown that a full stretching of native carbonic anhydrase B (CAB) using the atomic force microscope could not be achieved, presumably due to the presence of a 'knot' in the C-terminal region of the protein. When we used an engineered dimer of CAB, where the N-terminal monomeric unit (unit I) was expected to be 'knotless', we successfully recorded extension of the protein up to 110 nm which was long enough to account for the full extension of unit I monomer. In this paper we report that, by limiting the maximum length of extension to 90 nm extensions (corresponding to about 80 nm extension of the dimer and 70 nm of unit I), retractions of the polypeptide chain can be repeated cyclically without breaking the covalent crosslinking system. The force-extension curves obtained from the forward and reverse cycles of such experiments were almost perfectly superimposable with each other and with the corresponding part of the curves obtained from full extension experiments suggesting that the structure of unit I in the dimer was reversibly stretched and contracted. During the stretching of unit I of the dimer in either type of the experiments mentioned above, we occasionally observed a force peak having the force of about 0.5-0.7 nN when extension length reached 40-50 nm. We interpreted the appearance of such force peaks as an indication of formation of a tightly folded domain structure in unit I of CAB dimer.