Reduction of salt-requirement of halophilic nucleoside diphosphate kinase by engineering S-S bond.

Nucleoside diphosphate kinase (HsNDK) from extremely halophilic haloarchaeon, Halobacterium salinarum, requires salt at high concentrations for folding. A D148C mutant, in which Asp148 was replaced with Cys, was designed to enhance stability and folding in low salt solution by S-S bond. It showed increased thermal stability by about 10 °C in 0.2 M NaCl over the wild type HsNDK. It refolded from heat-denaturation even in 0.1 M NaCl, while the wild type required 2 M NaCl to achieve the same level of activity recovery. This enhanced refolding is due to the three S-S bonds between two basic dimeric units in the hexameric HsNDK structure, indicating that assembly of the dimeric unit may be the rate-limiting step in low salt solution. Circular dichroism and native-PAGE analysis showed that heat-denatured HsNDK formed partially folded dimeric structure, upon refolding, in the absence of salt and the native-like secondary structure in the presence of salt above 0.1 M NaCl. However, it remained dimeric upon prolonged incubation at this salt concentration. In contrary, heat-denatured D148C mutant refolded into tetrameric folding intermediate in the absence of salt and native-like structure above 0.1 M salt. This native-like structure was then converted to the native hexamer with time.