Critical Beginnings: Selective Tuning of Solubility and Structural Accuracy of Newly Synthesized Proteins by the Hsp70 Chaperone System

Abstract

Proteins are particularly prone to aggregation immediately after release from the ribosome, and it is therefore important to elucidate the role of chaperones during these key steps of protein life. The Hsp70 and trigger factor (TF) chaperone systems interact with nascent proteins during biogenesis and immediately post-translationally. It is unclear, however, whether these chaperones can prevent formation of soluble and insoluble aggregates. Here, we address this question by monitoring the solubility and structural accuracy of globin proteins biosynthesized in an Escherichia coli cell-free system containing different concentrations of the bacterial Hsp70 and TF chaperones. We find that Hsp70 concentrations required to grant solubility to newly synthesized proteins are extremely sensitive to client-protein sequence. Importantly, Hsp70 concentrations yielding soluble client proteins are insufficient to prevent formation of soluble aggregates. In fact, for some aggregation-prone protein variants, avoidance of soluble-aggregate formation demands Hsp70 concentrations that exceed cellular levels in E. coli. In all, our data highlight the prominent role of soluble aggregates upon nascentprotein release from the ribosome and show the limitations of the Hsp70 chaperone system in the case of highly aggregation-prone proteins. These results demonstrate the need to devise better strategies to prevent soluble-aggregate formation upon release from the ribosome.

Mapping Protein–Protein Interactions at Birth: Single-Particle Cryo-EM Analysis of a Ribosome–Nascent Globin Complex

“…These proteins include dihydrofolate reductase (DHFR), λ-lysozyme, green fluorescent protein, firefly luciferase, , tailspike, and several others . Additional recent work on apoMb revealed that the ribosome has a nascent chain solubilizing character. , The above results show that the ribosome has a remarkable ability to support the solubility and folding of a variety of proteins even in the complete absence of external molecular chaperones. The findings presented above, in combination with the research presented here, suggest a link between the nonpolar character of the newly discovered L23 interaction site and the known nascent chain solubilizing action of the ribosome.…”

“…On the other hand, translation outcomes of wild-type apoMb are very similar in the absence and presence of TF, with the only detectable difference being the presence of ca. 30% fewer soluble aggregates when TF is present . Previous studies showed that the interaction of full-length nascent E. coli globin domains with ribosomal proteins are mostly displaced by interactions with TF at physiologically relevant TF concentrations (ca.…”

“…The ribosome has also been found to increase nascent chain solubility , and to either destabilize or stabilize nascent chains relative to their native state. Further, the compact domains found in connection with the cotranslational folding of single-domain proteins are extremely spatially biased ,,− by bearing small-amplitude motions and are characterized by higher conformational dynamics than ribosome-released native states. In the case of intrinsically disordered nascent proteins, no compact regions are observed in any tunnel or outer ribosomal region, yet the ribosome participates in the process by severely limiting nascent chain conformational dynamics relative to ribosome-released proteins. − …”

“…The exit tunnel vestibule and nearby outer ribosomal surface, on the other hand, are wider than the tunnel core and more generally support nascent chain conformational sampling, leading to tertiary structure formation. In the case of small- to medium-size single-domain proteins, these areas of the ribosome host compact nascent-chain subdomains − that bear a native-like tertiary structure . Alternatively, in case the nascent chain bearing a C-terminal linker spanning ca.…”

“…7–35 residues, entire folded-like domains can be hosted. − The C-terminal linkers enable most (or all) residues comprising the protein domain to interact with each other and generate the native state within the vestibule and upper regions of the tunnel. The ribosome has also been found to increase nascent chain solubility , and to either destabilize or stabilize nascent chains relative to their native state. Further, the compact domains found in connection with the cotranslational folding of single-domain proteins are extremely spatially biased ,,− by bearing small-amplitude motions and are characterized by higher conformational dynamics than ribosome-released native states.…”