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Energetic dependencies dictate folding mechanism in a complex protein
Author(s) -
Kaiser Christian,
Chen Xiuqi,
Liu Kaixian
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.00621
Subject(s) - protein folding , folding (dsp implementation) , biophysics , ribosome , chemistry , protein domain , translation (biology) , population , crystallography , biology , biochemistry , rna , demography , sociology , messenger rna , gene , electrical engineering , engineering
Large proteins composed of multiple domains are abundant in all proteomes, but their folding and structural dynamics remain poorly understood. Multi‐domain proteins are thought to fold sequentially as they emerge from the ribosome, which minimizes interdomain misfolding. Once folded, domains interact with each other through the formation of extensive interfaces that are important for protein stability and function. In elongation factor G (EF‐G), a highly conserved protein composed of five domains, the two N‐terminal domains form a stably structured unit co‐translationally. Using single‐molecule optical tweezers, we have defined the steps leading to fully folded EF‐G. We find that the central domain III of EF‐G is highly dynamic and does not fold upon emerging from the ribosome. Surprisingly, a large interface with the N‐terminal domains does not contribute to the stability of domain III. Instead, it requires interactions with its folded C‐terminal neighbors to be stably structured. Because of the directionality of protein synthesis, this energetic dependency of domain III on its C‐terminal neighbors disrupts co‐translational folding and imposes a post‐translational mechanism on the folding of the C‐terminal part of EF‐G. As a consequence, unfolded domains accumulate during synthesis, leading to the extensive population of misfolded species that interfere with productive folding. Domain III flexibility enables large‐scale conformational transitions that are part of the EF‐G functional cycle during ribosome translocation. Our results suggest that energetic tuning of domain stabilities, which is likely crucial for EF‐G function, complicates the folding of this large multi‐domain protein. EF‐G thus provides an example of how distinct biological ends – robust folding and functionally important flexibility – come into conflict during protein biogenesis. Support or Funding Information This work was supported by a grant from the National Institutes of Health (5R01GM121567)Coupled synthesis and folding of the multi‐domain protein EF‐G. Domain III (green) is highly dynamic in the absence of fully folded domains IV (blue) and V (purple). As a consequence, co‐translational folding is interrupted. Accumulation of unfolded polypeptide results in the formation of misfolded species, sidetracking the molecule into non‐productive states and slowing down folding.