Co-translational Binding of GroEL to Nascent Polypeptides Is Followed by Post-translational Encapsulation by GroES to Mediate Protein Folding

Abstract

The eubacterial chaperonins GroEL and GroES are essential chaperones and primarily assist protein folding in the cell. Although the molecular mechanism of the GroEL system has been examined previously, the mechanism by which GroEL and GroES assist folding of nascent polypeptides during translation is still poorly understood. We previously demonstrated a cotranslational involvement of the Escherichia coli GroEL in folding of newly synthesized polypeptides using a reconstituted cellfree translation system (Ying, B. W., Taguchi, H., Kondo, M., and Ueda, T. (2005) J. Biol. Chem. 280, 12035-12040). Employing the same system here, we further characterized the mechanism by which GroEL assists folding of translated proteins via encapsulation into the GroEL-GroES cavity. The stable co-translational association between GroEL and the newly synthesized polypeptide is dependent on the length of the nascent chain. Furthermore, GroES is capable of interacting with the GroELnascent peptide-ribosome complex, and experiments using a single-ring variant of GroEL clearly indicate that GroES association occurs only at the trans-ring, not the cis-ring, of GroEL. GroEL holds the nascent chain on the ribosome in a polypeptide length-dependent manner and post-translationally encapsulates the polypeptide using the GroES cap to accomplish the chaperonin-mediated folding process.The Escherichia coli chaperonin GroEL is an essential heat shock protein, reaching ϳ1% of total cytoplasmic proteins (1-5). GroEL supports the folding of a considerable number of proteins with the assistance of the co-chaperonin GroES (6 -11). GroEL forms a large ring-shaped structure comprised of two heptameric rings of identical 57-kDa subunits, and these rings are stacked back-to-back (12, 13).GroEL executes two consecutive processes: binding of substrate proteins to prevent irreversible aggregation (the holder function) and release of the arrested protein to complete folding (the folder function) (5). The orderly progression of these two reactions is coordinated by ATP and GroES as follows. The substrate polypeptide is bound through multiple apical domains (7) along the inside surface of the GroEL rings (14). ATP-triggered binding of GroES to the substrate-loaded GroEL cis-ring leads to a release of the substrate protein. In the most productive pathway, the release of the substrate protein results in its encapsulation in a cavity formed by the GroEL-GroES complex (15-17). Both GroES and the substrate protein contact the cis-ring of GroEL but are subsequently detached by the binding of ATP to the trans-ring (18).The mechanistic details of GroEL function in the folding of chemically denatured proteins have been well elucidated (1-5). In E. coli, three major chaperone systems, the trigger factor (TF), 3 DnaK, and GroEL systems, are known to be involved in the folding of translated proteins (4, 19). The prevailing view assumes that TF and the DnaK system act as co-translational chaperones to prevent protein aggregation, whereas GroEL acts as a post-translational ch...

Improved Cell-Free RNA and Protein Synthesis System

“…Chaperone systems such as DnaK/DnaJ/GrpE and GroEL/GroES are understood to contribute to the folding of newly synthesized polypeptides, either interacting with these at the ribosome or shortly after their release [26], [28], [34]. Niwa etc.…”

“…reported that ribosome recycling times are minimized when RF1 concentrations are slightly smaller than the total ribosome concentration in an in vitro translation system [23]. Chaperone systems (e.g., GroEL/ES and DnaK/DnaJ/GrpE) were shown to alleviate protein aggregation when added to the PURE system [26]–[28], but their effects on the yield of different functional proteins are yet to be characterized. Recently identified Elongation Factor 4 (EF4), which induces back-translocations in ribosomes that have experienced defective translocations, was also shown to affect E. coli crude extract CFPS productivity [29].…”

Protein folding and aggregation in bacteria

“…In this configuration, GroEL holds the nascent polypeptide chain on the ribosome in a length-dependent manner and the protein is post-translationally encapsulated by the binding of the GroES cap to initiate the above-described chaperonin-assisted folding process [99], a process that again links protein synthesis to protein folding. (IV) ATP and GroES rebind to the trans GroEL ring, promoting a conformational change on the cis GroEL ring and liberating the polypeptide into the solution.…”

Exact methods for lattice protein models

“…It was shown through refolding experiments that the functional fold of a protein is mainly encoded by its sequence of amino acids [2,3]. Nevertheless, there is a large number of processes, like crowding effects [4], co-factor binding [5], or chaperons [6], that influence or support the structure formation process within living cells [7]. Still, the folding process is mainly governed by non-covalent intramolecular interactions [8] and misfolding can result in reduced or broken functionality [9].…”