A stable cold folding intermediate of rabbit muscle D-glyceraldehyde 3-phosphate dehydrogenase

With decreasing temperature the reactivation yield of denatured D-glycerald ehyde 3-phosphate dehydrogenase (GAPDH) upon dilution increases but the rea ctivation rare decreases. Neither reactivation nor aggregation during refol ding can be detected at 4 degrees C in 48 h, and at 3 degrees C even in 6 d ays. However, the reactivation takes place once the temperature is raised w ith little decrease of the yield after incubation for 6 days at 3 degrees C . A cold folding intermediate forms in a burst phase of refolding at 4 degr ees C as shown by a fast change of the intrinsic fluorescence followed by f urther conformational adjustment to a stable state in about 1 h. The stable folding intermediate has been characterized to be a dimer of partially fol ded GAPDH subunit with secondary structure between that of the native and d enatured enzymes, a hydrophobic cluster not found in either the native or t he denatured state, and an active site similar to but different from that o f the native stare. Chaperonin 60 (GroEL) binds with all intermediates form ed at 4 degrees C, but the intermediates formed at the early folding stage reactivate with higher yield than those formed after conformational adjustm ent when dissociated from GroEL in the presence of ATP and further folded a nd assembled into the,native tetramer.