Quasi-continuous cotranslational compaction and folding of a multidomain protein

Abstract

Most proteins start to fold cotranslationally as they come off the ribosome. So far, studies of cotranslational folding have focused mainly on small, single-domain proteins. Here, we have used Force Profile Analysis to study the cotranslational folding of Firefly Luciferase, a complex 550-residue protein composed of an N-terminal domain (NTD) encompassing two split Rossmann folds (RF-1, RF-2) and a β-roll, and a flexibly attached C-terminal domain (CTD). The folding process is characterized by a quasi-continuous series of compaction/folding steps that generate intermediate-size pulling forces on the nascent chain, punctuated by a prominent high-force event that represents the folding of the RF-2 domain, and a few low-force instances that likely indicate the formation of distinct folding intermediates. Trigger Factor interacts extensively with the nascent chain when the central part of RF-2 and the early parts of the CTD are synthesized. Our analysis uncovers a cotranslational compaction/folding process that is rich in detail and not just a simple succession of a few distinct, cooperative folding transitions. Force Profile Analysis show that firefly luciferase folds co-translationally through a continuous compaction process, punctuated by a major subdomain folding event. Trigger Factor binds to the nascent chain at distinct steps in the folding profile.