Energetic dependencies dictate folding mechanism in a complex protein

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Large multidomain proteins often fold cotranslationally to prevent misfolding.
• Domain interactions are crucial for protein stability and function.
• The folding pathways and energetics of multidomain proteins remain largely unknown.

Purpose of the Study:

• To investigate the folding process of the multidomain protein elongation factor G (EF-G).
• To understand the energetics of domain interactions within EF-G.
• To elucidate the mechanisms governing EF-G folding and its functional implications.

Main Methods:

• Single-molecule optical tweezers to monitor folding steps.
• Analysis of domain stability and interactions in EF-G.

Main Results:

• The N-terminal domains of EF-G fold cotranslationally into a stable unit.
• Domain III of EF-G is highly dynamic and does not fold immediately after synthesis.
• Domain III stability depends on interactions with C-terminal domains, imposing a posttranslational folding requirement.
• Accumulation of unfolded domains leads to misfolded species, potentially affecting EF-G function.
• Domain III flexibility is essential for EF-G's role in ribosome translocation.

Conclusions:

• EF-G folding is not entirely cotranslational, with a significant posttranslational component.
• The energetic tuning of domain stabilities complicates EF-G folding but is likely critical for its function.
• Understanding EF-G folding provides insights into the challenges of large multidomain protein biogenesis.