Systematic mapping of the free energy landscapes of a growing immunoglobulin domain identifies a kinetic intermediate associated with co-translational proline isomerization

Co-translational folding is a fundamental molecular process that ensures efficient protein biosynthesis and minimizes the wasteful or hazardous formation of misfolded states. However, the complexity of this process makes it extremely challenging to obtain structural characterizations of co-translational folding pathways. Here we contrast observations in translationally-arrested nascent chains with those of a systematic C-terminal truncation strategy. We create a detailed description of chain length-dependent free energy landscapes associated with folding of the FLN5 filamin domain, in isolation and on the ribosome. By using this approach we identify and characterize two folding intermediates, including a partially folded intermediate associated with the isomerization of a conserved proline residue, which, together with measurements of folding kinetics, raises the prospect that neighboring unfolded domains might accumulate during biosynthesis. We develop a simple model to quantify the risk of misfolding in this situation, and show that catalysis of folding by peptidyl-prolyl isomerases is essential to eliminate this hazard.