Function of Trigger Factor and DnaK in Multidomain Protein Folding

Abstract

Trigger factor and DnaK protect nascent protein chains from misfolding and aggregation in the E. coli cytosol, but how these chaperones affect the mechanism of de novo protein folding is not yet understood. Upon expression under chaperone-depleted conditions, multidomain proteins such as bacterial beta-galactosidase (beta-gal) and eukaryotic luciferase fold by a rapid but inefficient default pathway, tightly coupled to translation. Trigger factor and DnaK improve the folding yield of these proteins but markedly delay the folding process both in vivo and in vitro. This effect requires the dynamic recruitment of additional trigger factor molecules to translating ribosomes. While beta-galactosidase uses this chaperone mechanism effectively, luciferase folding in E. coli remains inefficient. The efficient cotranslational domain folding of luciferase observed in the eukaryotic system is not compatible with the bacterial chaperone system. These findings suggest important differences in the coupling of translation and folding between bacterial and eukaryotic cells.

Structure and dynamics of a ribosome-bound nascent chain by NMR spectroscopy

“…This result is significant because it shows the presence of a highly folded structure of the nascent Ig2 NTD even when attached to the ribosome (Fig. 2) (26,27). The molecular chaperone, trigger factor, could not be identified in the purified RNCs (see SI Fig.…”

Structure of trigger factor binding domain in biologically homologous complex with eubacterial ribosome reveals its chaperone action

“…Furthermore, TF is known to delay the folding and͞or misfolding of nascent chains by recognizing hydrophobic regions in nascent polypeptides (25). The altered conformation of the TFa binding region forces helices A1 and A3 to lie further apart from one another, and consequently separates helix A3 from the ␤-sheets, thereby exposing a substantial hydrophobic pocket facing the opening of the ribosomal tunnel.…”

“…In cases of multidomain bacterial proteins, such as ␤-galactosidase, additional TF molecules are recruited during translation, suggesting that, while maintaining contact with the elongated chain, the initially bound TF leaves the ribosomal docking site once sufficient sequence information is available for the generation of a folded core (25). However, because the dissociation of TF from the ribosome is a slow process (32), and protein domains vary in size and structure, a dynamic control of TF dissociation and reassociation during the translation of multidomain proteins is necessary to maintain efficient chaperone activity.…”

Firefly luciferase mutants as sensors of proteome stress

“…In eukaryotes, a polypeptide can begin to fold to form a protein domain as soon as it emerges from the ribosomes, a process called co-translational folding. However, a domain can only completely fold when its entire sequence has emerged from the ribosome (Agashe et al, 2004;Netzer and Hartl, 1997). This scenario exposes non-native surfaces on nascent chains for a considerable length of time to interact non-specifically and make them highly aggregation prone.…”

“…Ribosome Associated Chaperones translational folding (Agashe et al, 2004;Kaiser et al, 2006;Kramer et al, 2009). This provides sufficient time to the elongating polypeptide to receive its entire structural information requisite for the productive folding to begin.…”