Folding and assembly of co-chaperonin heptamer probed by forster resonance energy transfer.

The ring-shaped heptameric co-chaperonin protein 10 (cpn10) is one of few oligomeric beta-sheet proteins that unfold and disassemble reversibly in vitro. Here, we labeled human mitochondrial cpn10 with donor and acceptor dyes to obtain FRET signals. Cpn10 mixed in a 1:1:5 ratio of donor:acceptor:unlabeled monomers form heptamers that are active in an in vitro functional assay. Monomer-monomer affinity, as well as thermal and chemical stability, of the labeled cpn10 is similar to the unlabeled protein, demonstrating that the labels do not perturb the system. Using changes in FRET, we then probed for the first time cpn10 heptamer-monomer assembly/disassembly kinetics. Heptamer dissociation is very slow (1/k(diss) approximately 3h; 20 degrees C, pH 7) corresponding to an activation energy of approximately 50kJ/mol. Ring-ring mixing experiments reveal that cpn10 heptamer dissociation is rate limiting; subsequent associations events are faster. Kinetic inertness explains how cpn10 cycles on and off cpn60 as an intact heptamer in vivo.