THERMODYNAMIC STABILITY AND FOLDING OF GROEL MINICHAPERONES

The apical domain of GroEL (residues 191 to 376) and its C-terminally truncated fragment GroEL(191-345) are expressed with high yield in Esc herichia coli to give functional monomeric minichaperones. Owing to th e reversible folding behaviour of the minichaperones we can analyse th e folding of the polypeptide binding domain of the multidomain GroEL p rotein, the folding of which is known to be irreversible. The apical d omain shows two reversible temperature transitions with transition mid points at 35 degrees C and at 67 degrees C that can be attributed to t he unfolding of the C-terminal helices and the domain core, respective ly. The native state of the domain core is stabilized by 5.5 kcal mol( -1) relative to the unfolded state. The rate constant of folding of th e apical domain core is independent of the minichaperone concentration and the presence of the C-terminal alpha-helices. A folding intermedi ate on the folding pathway is destabilized relative to the native stat e by 1.6 kcal mol(-1), which is also detected by equilibrium and kinet ic binding of the dye bis-ANS. Reversible folding of the polypeptide d omain of GroEL guarantees highly efficient chaperonin activity within the GroEL toroid. (C) 1998 Academic Press Limited.