GROEL-MEDIATED FOLDING OF STRUCTURALLY HOMOLOGOUS DIHYDROFOLATE REDUCTASES

Using stopped-flow fluorescence techniques, we have examined both the refolding and unfolding reactions of four structurally homologous dihy drofolate, reductases (murine DHFR, wild-type E. coli DHFR, and two E. coli DHFR mutants) in the presence and absence of the molecular chape ronin GroEL, We show that GroEL binds the unfolded conformation of eac h DHFR with second order rate constants greater than 3 x 10(7) M(-1)s( -1) at 22 degrees C. Once bound to GroEL, the proteins refold with rat e constants similar to those for folding in the absence of GroEL, The overall rate of formation of native enzyme is decreased by the stabili ty of the complex between GroEL and the last folding intermediate. For wild-type E. coli DHFR, complex formation is transient while for the others, a stable complex is formed. The stable complexes are the same regardless of whether they are formed from the unfolded or folded DHFR . When complex formation is initiated from the native conformation, Gr oEL binds to a pre-existing non-native conformation, presumably a late folding Intermediate, rather than to the native state, thus; shifting the conformational equilibrium toward the non-native species by mass action. The model presented here for the interaction of these four pro teins with GroEL quantitatively describes the difference between the f ormation of a transient complex and a stable complex as defined by the rate constants for release and rebinding to GroEL relative to the rat e constant for the last folding step. Due to this kinetic partitioning , three different mechanisms can be proposed for the formation of stab le complexes between GroEL and either murine DHFR or the two E. coli D HFR mutants. These data show that productive folding of GroEL-bound pr oteins can occur in the absence of nucleotides or the co-chaperonin Gr oEL and suggest that transient complex formation may be the functional role of GroEL under normal conditions. (C) 1997 Academic Press Limite d.