Analysis of GroE-assisted Folding under Nonpermissive Conditions

Holger Grallert,Johannes Buchner

doi:10.1074/jbc.274.29.20171

Holger Grallert, Johannes Buchner

Open Access

https://doi.org/10.1074/jbc.274.29.20171

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Abstract

The molecular chaperones GroEL and GroES facilitate protein folding in an ATP-dependent manner under conditions where no spontaneous folding occurs. It has remained unknown whether GroE achieves this by a passive sequestration of protein inside the GroE cavity or by changing the folding pathway of a protein. Here we used citrate synthase, a well studied model substrate, to discriminate between these possibilities. We demonstrate that GroE maintains unfolding intermediates in a state that allows productive folding under nonpermissive conditions. During encapsulation of non-native protein inside GroEL.GroES complexes, a folding reaction takes place, generating association-competent monomeric intermediates that are no longer recognized by GroEL. Thus, GroE shifts folding intermediates to a productive folding pathway under heat shock conditions where even the native protein unfolds in the absence of GroE.

Highlights

Molecular chaperones are known to play a major role in protein folding in the cell
Folding in cis-complexes is restricted by the size of the central cavity of the GroEL1⁄7GroES complexes to polypeptides smaller than 60 kDa [3, 30]
Influence of the GroE System on the Thermal Unfolding of CS—In the presence of GroEL, the native CS dimer unfolds thermally via inactive dimeric intermediates, which dissociate into monomers

Summary

Introduction

Molecular chaperones are known to play a major role in protein folding in the cell. One of the best characterized chaperones is the GroEL/GroES system from Escherichia coli. The GroE chaperones maintain viability by stabilizing unfolding proteins or by keeping unfolding intermediates in a reactivable state and preventing irreversible side reactions like aggregation (4 –7). This allows refolding of the bound intermediates after restoration of permissive folding conditions. The presence of GroES, the heptameric ring-shaped co-chaperone of GroEL, together with ATP is required for increasing the efficiency of substrate folding and for folding under nonpermissive conditions [22, 23] This seems to be due to the ability of GroE to partially unfold kinetically trapped folding intermediates, giving these species a new chance to fold [23,24,25]. Shifting of monomeric CS intermediates from the unfolding pathway to an alternative folding pathway ensures that active protein is formed in the cell even under unfavorable environmental conditions

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