Though in vitro studies have produced insight into mechanisms underlying protein folding, determining how cellular factors modulate folding remains challenging. Proteins have the opportunity to fold when synthesized from N- to C-terminus by the ribosome at a rate of around five to ten residues per second in cells. This is much slower than the average folding rate of most small proteins, giving the nascent chain time to sample many conformations during translation. When refolding in vitro, HaloTag populates an aggregation-prone intermediate that contains structure from both the N- and C-termini, but this intermediate does not form co-translationally. Could altering the chain connectivity of HaloTag decrease its folding efficiency? We have shown how changing the vectorial synthesis of HaloTag through circular permutation affects folding efficiency in vitro and in vivo. We assessed all 298 circular permutants (CPs) of HaloTag in E. coli cell lysate and showed that circular permutation positions differentially affect protein solubility and function. FACS experiments in live mammalian HEK293T cells demonstrate that many CPs retain function in an alternative cellular environment. Combining these data with both standard in vitro protein folding assays and hydrogen-exchange MS studies to map the folding process yields insight into how circular permutation alters the folding landscape of HaloTag and the role of co-translational folding in this process.

Comparing protein folding on and off the ribosome

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