Cooperativity of α- and β-Subunits of Group II Chaperonin from the Hyperthermophilic Archaeum Aeropyrum pernix K1

Abstract

alpha- and beta-subunits (ApCpnA and ApCpnB) are group II chaperonins from the hyperthermophilic archaeum Aeropyrum pernix K1, specialized in preventing the aggregation and inactivation of substrate proteins under conditions of transient heat stress. In the present study, the cooperativity of alpha- and beta-subunits from the A. pernix K1 was investigated. The ApCpnA and ApCpnB chaperonin genes were overexpressed in E. coli Rosetta and Codonplus (DE3), respectively. Each of the recombinant alpha- and beta- subunits was purified to 92% and 94% by using anionexchange chromatography. The cooperative activity between purified alpha- and beta-subunits was examined using citrate synthase (CS), alcohol dehydrogenase (ADH), and malate dehydrogenase (MDH) as substrate proteins. The addition of both alpha- and beta-subunits could effectively protect CS and ADH from thermal aggregation and inactivation at 43 degreesC and 50 degreesC, respectively, and MDH from thermal inactivation at 80 degreesC and 85 degreesC. Moreover, in the presence of ATP, the protective effects of alpha- and beta-subunits on CS from thermal aggregation and inactivation, and ADH from thermal aggregation, were more enhanced, whereas cooperation between chaperonins and ATP in protection activity on ADH and MDH (at 85 degreesC) from thermal inactivation was not observed. Specifically, the presence of both alpha- and beta- subunits could effectively protect MDH from thermal inactivation at 80 degreesC in an ATP-dependent manner.