Chaperonin-mediated folding in the eukaryotic cytosol proceeds through rounds of release of native and nonnative forms.

The eukaryotic cytosolic chaperonin, CCT, plays an essential role in mediating ATP-dependent folding of actin and tubulin. There is debate about whether it mediates folding through a single round of association followed by release of native forms, or through cycles of binding and full release in which only a fraction of released molecules reaches native form in any cycle. We examine the fate of newly synthesized substrate proteins bound to CCT in reticulocyte lysate or intact Xenopus oocytes. When a chaperonin "trap," able to bind but not release substrate protein, is introduced, production of the native state is strongly inhibited, associated with transfer to trap. While predominantly nonnative forms of actin, tubulin, and a newly identified substrate, G(alpha)-transducin, are released from CCT, a small fraction reaches native form with each round of release, inaccessible to trap. This overall mechanism resembles that of the bacterial chaperonin, GroEL.

Native-like structure of a protein-folding intermediate bound to the chaperonin GroEL.

The chaperonin GroEL binds nonnative proteins in its central channel through hydrophobic interactions and initiates productive folding in this space underneath bound co-chaperone, GroES, in the presence of ATP. The questions of where along the folding pathway a protein is recognized by GroEL, and how much structure is present in a bound substrate have remained subjects of discussion, with some experiments suggesting that bound forms are fully unfolded and others suggesting that bound species are partially structured. Here we have studied a substrate protein, human dihydrofolate reductase (DHFR), observing in stopped-flow fluorescence experiments that it can rapidly bind to GroEL at various stages of folding. We have also analyzed the structure of the GroEL-bound protein using hydrogen-deuterium exchange and NMR spectroscopy. The pattern and magnitude of amide proton protection indicate that the central parallel beta-sheet found in native DHFR is present in a moderately stable state in GroEL-bound DHFR. Considering that the strands are derived from distant parts of the primary structure, this suggests that a native-like global topology is also present. We conclude that significant native-like structure is present in protein-folding intermediates bound to GroEL.