Cotranslational Folding Promotes β-Helix Formation and Avoids Aggregation In Vivo

Abstract

Newly synthesized proteins must form their native structures in the crowded environment of the cell, while avoiding non-native conformations that can lead to aggregation. Yet remarkably little is known about the progressive folding of polypeptide chains during chain synthesis by the ribosome, or of the influence of this folding environment on productive folding in vivo. P22 tailspike is a homotrimeric protein that is prone to aggregation via misfolding of its central β-helix domain in vitro. We have produced stalled ribosome:tailspike nascent chain complexes of four fixed lengths in vivo, in order to assess co-translational folding of newly synthesized tailspike chains as a function of chain length. Partially synthesized, ribosome-bound nascent tailspike chains populate stable conformations with some native-state structural features even prior to the appearance of the entire β-helix domain, regardless of the presence of the chaperone trigger factor, yet these conformations are distinct from the conformations of released, refolded tailspike truncations. These results suggest that organization of the aggregation-prone β-helix domain occurs co-translationally, prior to chain release, to a conformation that is distinct from the accessible energy minimum conformation for the truncated free chain in solution.As a protein is synthesized by the ribosome, it begins to fold into a three-dimensional shape, and must simultaneously avoid aggregation with other proteins in the crowded cell. In this seemingly hostile environment, where total protein concentrations can exceed 200-300 mg/ mL, de novo protein folding during and after translation is, for many proteins, more efficient than in vitro refolding1. In other words, many proteins that can fold productively in the cell will aggregate severely under in vitro refolding reactions, presumably due to differences between the dominant folding pathway used. For example, experiments with both bacterial and firefly luciferase have demonstrated that these nascent chains adopt conformations cotranslationally that are not populated during refolding from denaturant2,3, and these cotranslational conformations fold to the native state much more efficiently than the conformations populated during refolding from denaturant. However, there continues to be debate as to what extent large, multi-domain proteins can fold co-translationally in prokaryotes4-7.While molecular chaperones certainly play a role in efficient protein folding in the cell, less than 20% of E. coli cytoplasmic proteins require an interaction with one of the three major chaperone systems (trigger factor (TF), DnaK/DnaJ, or GroEL/ES) in order to fold correctly under normal growth conditions8. And remarkably, both chaperone systems responsible for Correspondence should be addressed to P.L.C. (pclark1@nd.edu). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript wil...

The ribosome regulates flavodoxin folding

“…We note that the apo form of -5 P is more susceptible to degradation than apo-FL P , which is in agreement with our observation that site-directed mutagenesis of apoflavodoxin decreases the stabilities of the corresponding native apo-proteins (38,45,51,52). In addition, flexibility of the flavin-binding site in flavodoxin is much lower than in apoflavodoxin and holo-protein is much more stable than apo-protein (25,53,54).…”

“…As FMN synthesis is relatively slow, it takes much longer to assemble all the protein in holo form than the time in which the maximum of apo-protein is synthesized (57). Due to amino acid residue replacement, F44Y, W128F, W167F or W128F/W167F apoflavodoxin variants are destabilized compared to wild-type protein (38,45,51,52). Thus, these apo-proteins are more efficiently degraded by intracellular proteases before they can take advantage of the stabilization conferred through binding of FMN.…”

“…Each construct (i.e., C69A or F44Y) was transformed in Escherichia coli (strain TG2) and purified as described previously (46). Apoprotein was prepared according to established protocols (52). Holo-and apoproteins were flash-frozen in liquid nitrogen and stored in KPPi buffer, pH 6.0, at -80 °C.…”

“…Such insertions often happen cotranslationally, i.e., while the ribosome synthesizes the polypeptide concerned. Proteins can fold cotranslationally and sample intermediate folding states (51)(52)(53)(54)(55)(56)(57), which might include MGs.…”

“…Depending on nascent chain conformation, the exit tunnel can hold between 30 (extended) to 70 (α-helical) amino acid residues. Folding or misfolding of the nascent chain may already start in the exit tunnel (151)(152)(153)(154) and in most cases commences while the nascent chain gradually emerges from it (51)(52)(53)55,(155)(156)(157)(158)(159)(160)(161)(162)(163). As synthesis of the nascent chain is orders of magnitude slower than it takes a protein to fold (i.e., seconds versus micro-to milliseconds), nascent chains can already sample parts of their conformational space before the next amino acid residue is added.…”