Insight into the conformation of protein folding intermediate(s) trapped by GroEL.

Many aspects of the mechanism by which the GroEL/ES chaperonins mediate protein folding are still unclear, including the amount of structure present in the substrate bound to GroEL. To address this issue we have analyzed the susceptibility to limited proteolysis and to alkylation of cysteine residues of mitochondrial aspartate aminotransferase (mAAT) bound to GroEL. Several regions of the N-terminal portion of GroEL-bound mAAT are highly susceptible to proteolysis, whereas a large core of about 200 residues containing the C-terminal half of the polypeptide chain is protected in the complex. This protection does not extend to the mAAT sulfhydryl groups which in the GroEL-mAAT complex have similar reactivity as in fully unfolded mAAT. These results suggest that the mAAT species bound to GroEL represent folding intermediates with a conformation that is substantially more disorganized than that of the native state. The N-terminal half of the molecule is more flexible and lies exposed at the mouth of the central cavity of GroEL. The more compact C-terminal section of mAAT, which contains residues located at the subunit interface in the native dimer, appears to be hidden in the central cavity of GroEL. Thus, the bulk of the interactions in the GroEL.mAAT complex seems to involve residues from the more compact C-terminal section of the substrate.