Inter‐Domain Interactions In Nascent Polypeptides Interfere With Productive Protein Folding

How large, multi‐domain proteins robustly fold into their native structures is not well understood. It is often assumed that individual domains fold independently, but experimental studies have largely been limited to the special case of tandem repeat proteins. We have used single‐molecule force spectroscopy to study the folding of elongation factor G (EF‐G), which is composed of five domains. Optical tweezers experiments show that full‐length EF‐G refolds inefficiently along highly complex folding pathways. To determine whether vectorial protein synthesis, by reducing complexity, enables efficient domain‐wise folding, we studied successively longer ribosome‐bound nascent polypeptides. The N‐terminal G‐domain forms a stable structure by itself and enables the otherwise unstable subsequent domain II to fold. When both domains fold simultaneously, they form a misfolded species that is more stable than the productive folding intermediates. Interestingly, interactions with the unfolded domain II destabilize the native G‐domain. Together, our data indicate that vectorial protein synthesis, perhaps with rates optimized for folding, helps to avoid intra‐molecular misfolding within nascent multi‐domain polypeptides, allowing the G‐domain to complete folding before domain II is produced. Interactions with molecular chaperones might be required to protect folded domains against interactions with yet unstructured regions in the nascent polypeptide. Support or Funding Information Johns Hopkins University, The Pew Charitable Trusts