Functional consequences of single:double ring transitions in chaperonins: life in the cold

• Published in: Molecular Microbiology
• Main Authors: Ferrer, Manuel, Lünsdorf, Heinrich, Chernikova, Tatyana N., Yakimov, Michail, Timmis, Kenneth N., Golyshin, Peter N.
• Format: Article in Journal/Newspaper
• Language: English
• Published: Wiley 2004
• Subjects: Antarc*; Antarctica
• Online Access: http://dx.doi.org/10.1111/j.1365-2958.2004.04077.x; https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1111%2Fj.1365-2958.2004.04077.x; https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2958.2004.04077.x

Summary The cpn 60 and cpn 10 genes from psychrophilic bacterium, Oleispira antarctica RB8, showed a positive effect in Escherichia coli growth at low temperature, shifting its theoretical minimal growth temperature from +7.5°C to −13.7°C [Ferrer, M., Chernikova, T.N., Yakimov, M., Golyshin, P.N., and Timmis, K.N. (2003) Nature Biotechnol 21: 1266–1267]. To provide experimental support for this finding, Cpn60 and 10 were overproduced in E. coli and purified to apparent homogeneity. Recombinant O.Cpn60 was identical to the native protein based on tetradecameric structure, and it dissociates during native PAGE. Gel filtration and native PAGE revealed that, in vivo and in vitro , (O.Cpn60) 7 was the active oligomer at 4–10°C, whereas at > 10°C, this complex was converted to (O.Cpn60) 14 . The dissociation reduces the ATP consumption (energy‐saving mechanism) and increases the refolding capacity at low temperatures. In order for this transition to occur, we demonstrated that K468 and S471 may play a key role in conforming the more advantageous oligomeric state in O.Cpn60. We have proved this hypothesis by showing that single and double mutations in K468 and S471 for T and G, as in E.GroEL, produced a more stable double‐ring oligomer. The optimum temperature for ATPase and chaperone activity for the wild‐type chaperonin was 24–28°C and 4–18°C, whereas that for the mutants was 45–55°C and 14–36°C respectively. The temperature inducing unfolding (T M ) increased from 45°C to more than 65°C. In contrast, a single ring mutant, O.Cpn60 SR , with three amino acid substitutions (E461A, S463A and V464A) was as stable as the wild type but possessed refolding activity below 10°C. Above 10°C, this complex lost refolding capacity to the detriment of the double ring, which was not an efficient chaperone at 4°C as the single ring variant. We demonstrated that expression of O.Cpn60 WT and O.Cpn60 SR leads to a higher growth of E. coli at 4°C (µ max , 0.22 and 0.36 h −1 respectively), whereas at 10–15°C, only E. coli cells ...