Dramatic stabilization of the native state of human carbonic anhydrase II by an engineered disulfide bond.

To find a disulfide pair that could stabilize the enzyme human carbonic anhydrase II (HCA II), we grafted the disulfide bridge from the related and unusually stable carbonic anhydrase form from Neisseria gonorrhoeae(NGCA) into the human enzyme. Thus, the two Cys residues at positions 23 and 203 were engineered into a pseudo-wild-type form of HCA II (C206S), giving the mutant C206S/A23C/ L203C. The disulfide bond was not formed spontaneously. The native state of the reduced form of the mutant was markedly destabilized (2.9 kcal/mol) compared to that of HCA II. Formation of a disulfide bridge was achieved by treatment by oxidized glutathione. This led to a significant stabilization of the native conformation. Compared to HCA II the unfolding midpoint for the variant was increased from 0.9 to 1.7 M guanidine HCl, corresponding to a stabilization of 3.7 kcal/mol. This makes the human enzyme almost as stable as the model protein NGCA, for which the unfolding of the native state has a midpoint at 2.1 M guanidine HCl. The stabilized protein underwent, contrary to all other investigated variants of HCA II, an apparent two-state unfolding transition, as judged from intrinsic Trp fluorescence measurements. A molten-globule intermediate is nevertheless formed but is suppressed because of the high denaturant pressure it faces upon rupture of the native state. A major goal of protein engineering is the design of sta- bilized protein variants, and several strategies have been em- ployed to achieve this objective, for example, helix dipole stabilization (1, 2) and the classical method entailing intro- duction of novel disulfide bridges (reviewed in ref 3). How- ever, considering the latter approach, most attempts that have been successful have dealt with T4 lysozyme (4, 5) and ribo- nuclease H (6). Human carbonic anhydrase II (HCA II) is mainly a ‚-sheet protein that has 10 ‚-strands that divide the structure into two halves (Figure 1A). The upper part of the molecule con- sists of an N-terminal domain as well as a major domain that comprises the active site and the lower part of a large hydrophobic core. HCA II has been widely used as a model system for analysis of the catalytic (7), protein folding (8, 9), protein-chaperone (10), and protein-surface adsorption mechanisms (11). In the present study, our objective was to introduce a disulfide bridge into HCA II as a means of stabilizing the enzyme. To assign the bridge to suitable positions, we took advantage of the recently described structure of the enzyme