We have also on-going studies using step-wise mutation as well as DNA shuffling to understand the difference in stability between various carbonic anhydrases.
For one approach we are using CA from bacteria (N. gonorrhoeae; NGCA). Comparing the 3-D structure of NGCA and HCA II displays some differences. NGCA contains a disulfide bridge that is supposed to be a main contributor to the high stability of this enzyme. In an earlier project we have grafted the corresponding NGCA disulfide bridge into HCA II, yielding a remarkably stabilized mutant (Mårtensson et al (2002), Biochemistry 41, 15867-15875).
The inserted cysteine residues are placed in the interior of the structure and because of the conformationally restrained localization, as indicated from modeling, the protein is expressed in the reduced state and are not readily oxidized. However, in a recent work we have found that upon exposure to low concentrations of denaturant (0.6 M GuHCl) the oxidation rate of correctly formed disulfide bridges in the native state was markedly increased.
By entropy estimations it appears as it is the increased flexibility, induced by the denaturant, that enables the cysteines to find each other and hence to form the disulfide bridge (Karlsson et al ( 2005), Biochemistry, 44:3487-3493).
Figure: Overlay of a genetically engineered HCAII (blue) and carbonic anhydrase from Neisseria gonorrhoeae (red).
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Last updated: 05/27/08