Interaction of GroE with an all-β-protein

Molecular chaperones are involved in protein folding both in vivo and in vitro. The Escherichia coli chaperone GroEL interacts with a number of nonnative proteins. A common structural motif of nonnative proteins, which is recognized by GroEL, has not yet been identified. In order to study the role of β-sheet secondary structure on the interaction of nonnative proteins with GroEL, we used the F_ab fragment of a monoclonal antibody as a model substrate protein. Here we show that GroEL interacts functionally with this all-β-protein during reactivation. Antibody fragments refold spontaneously in good yield from the guanidine-denatured state. Functional refolding to the native state is inhibited transiently by GroEL, but there is no complete folding arrest in the absence of Mg-ATP and GroES. The yield of these unspecifically released GroEL-bound F_ab fragments corresponds to that of the spontaneous reactivation in the absence of chaperones. However, the refolding kinetics in the presence of GroEL are considerably slower. The addition of Mg-ATP to the GroEL·F_ab complex results in an immediate release of bound substrate protein and a significant increase in the amount of reconstituted antibody fragments compared to spontaneous reactivation GroES is not essential for functional GroEL-mediated refolding of the F_ab fragment but affects the reactivation yield to a small extent. Interestingly, stimulation of the GroEL-mediated F_ab refolding depends primarily on the binding and not on hydrolysis of adenosine triphosphates. Previous results indicate the binding of α-helices to GroEL. The results presented in this paper suggest that β-sheet secondary structural elements are recognized by GroEL. We therefore conclude that the interaction of a nonnative protein with GroEL depends mainly on the nature of the early folding intermediate but not on a specific element of secondary structure.