Testing mechanisms for homomeric protein assembly in vivo

Proper folding is a prerequisite for protein function. Most proteins are multimeric, meaning that folding includes the additional step of assembling individual subunits together to form the native multimeric structure. Although little is known about multimeric protein folding, failing to form proper subunit interactions can lead to loss of protein function, aggregation and/or degradation. During protein synthesis, individual subunits of homomeric proteins are close to one other on neighboring ribosomes and may start to assemble co-translationally (“co-co” assembly). Alternatively, nascent chains may interact co-translationally with a full-length subunit (“co-post” assembly). Work from the Bukau and Kramer labs indicates that co-co interactions between nascent chains enhances the efficiency of homomeric protein folding and assembly (Bertolini et al. (2021) Science). However, their approach was not capable of identifying the contribution of co-post assembly to efficient folding. Here we present results from a novel assay designed to test the extent to which co-post assembly occurs during homomultimeric protein assembly. As an initial model, we used the E. coli homotrimer chloramphenicol acetyltransferase (CAT). The CAT native trimer structure is thermostable to 80°C and shows no evidence of subunit exchange over the lifetime of E. coli. CAT does not refold to its native structure after dilution from a chemical denaturant, indicating that the “pioneer round” of folding, potentially including co-translational folding and/or assembly, is particularly important for achieving the native CAT structure. However, we and others have shown that CAT native structure formation requires the presence of the CAT C-terminal residues. To resolve this conundrum, we designed a novel fluorescence reporter assay to help distinguish between co-post versus other assembly mechanisms. Crucially, this assay should prove useful for testing the contribution of co-post assembly to the folding of any multimeric protein, including heteromeric multimers.