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

The cpn60 and cpn10 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 +7.5degreesC to -13.7degreesC [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 4-10degreesC, whereas at > 10degreesC, 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 24-28degreesC and 4-18degreesC, whereas that for the mutants was 45-55degreesC and 14-36degreesC respectively. The temperature inducing unfolding (T-M) increased from 45degreesC to more than 65degreesC. 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 10degreesC. Above 10degreesC, this complex lost refolding capacity to the detriment of the double ring, which was not an efficient chaperone at 4degreesC 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 4degreesC (mu(max), 0.22 and 0.36 h(-1) respectively), whereas at 10-15degreesC, only E. coli cells expressing O.Cpn60 or O.Cpn60(DR) grew better than parental cells (-cpn). These results clearly indicate that the single-to-double ring transition in Oleispira chaperonin is a wild-type mechanism for its thermal acclimation. Although previous studies have also reported single-to-double ring transitions under many circumstances, this is the first clear indication that single-ring chaperonins are necessary to support growth when the temperature falls from 37degreesC to 4degreesC.