GroES in the asymmetric GroEL(14)-GroES(7) complex exchanges via an associative mechanism

The interaction of the chaperonin GroEL(14) with its cochaperonin GroES(7) is dynamic, involving stable. asymmetric 1:1 complexes (GroES(7). GroEL(7)- GroEL(7)) and transient, metastable symmetric 2:1 complexes [GroES(7). GroE S(7)-GroEL(7). GroES(7)]. The transient formation of a 2:1 complex permits exchange of free GroES(7) for GroES(7) bound in the stable 1:1 complex, Ele ctrophoresis in the presence of ADP was used to resolve free GroEL(14) from the GroES(7)-GroEL(14) complex. Titration of GroEL(14) with radiolabeled G roES(7) to molar ratios of 32:I demonstrated a 1:1 Limiting stoichiometry i n a stable complex. No stable 2:1 complex was detected Preincubation of the asymmetric GroES(7). GroEL(7)-GroEL(7) complex with excess unlabeled GroES 7 in the presence of ADP demonstrated GroES(7) exchange, The rates of GroES (7) exchange were proportional to the concentration of unlabeled free GroES (7). This concentration dependence points to an associative mechanism in wh ich exchange of GroES(7) occurs by way of a transient 2:1 complex and exclu des a dissociative mechanism in which exchange occurs by way Of free GroEL( 14) Exchange of radiolabeled ADP from 1:1 complexes was much slower than th e exchange of GroES(7), In agreement with recent structural studies, this i ndicates that conformational changes in GroEL(14) following the dissociatio n of GroES(7) must precede ADP release, These results explain how the GroEL (14) cavity can become reversibly accessible to proteins under in vivo cond itions that favor 2:1 complexes.