Protein folding is a fundamental process that enables polypeptides to acquire their functional three-dimensional structures. However, the crowded environment inside the cell can cause protein misfolding by interfering with the proper folding process. A key question is when misfolding begins, and how proteins fold into their native structures. To better understand the details of how proteins fold into their native structure, we aim to uncover the structures of some intermediate states during co-translational protein folding on the ribosome by cryo-electron microscopy single-particle analysis (cryo-EM SPA) As a nascent chain (NC) model, we focused on Escherichia coli ( E. coli ) β-galactosidase for three reasons. (1) It undergoes co-translational folding, because it needs to form a tertiary structure on the ribosome. (2) Its molecular weight exceeds 50 kDa, making it easier to classify particles clearly during classification in cryo-EM analysis. (3) Its three-dimensional structure has already been determined so that the structural interpretation of the intermediates is facilitated. To sample different stages of β-gal synthesis, we generated a series of stalled ribosome-nascent chain complexes (RNCs) in E. coli by inserting an arrest ribosome stalling sequence (WWWPRIRGPPGS) upstream of the β-gal ORFs. Since the length of the C-terminal linker influences the conformational freedom of the nascent chain, we varied the length of the linker sequence and designed constructs to capture nascent chains starting within the ribosome exit tunnel, just emerging from the tunnel, or with the complete sequence of β-galactosidase exposed outside the exit tunnel. In this poster session, we report that ribosome-nascent chain complexes (RNCs) were visualized by expressing these plasmids in E. coli and analyzing them using cryo-EM SPA.
Konishi et al. (Sun,) studied this question.