Abstract
Protein folding in the cell often begins during translation. Many proteins fold more efficiently cotranslationally than when refolding from a denatured state. Changing the vectorial synthesis of the polypeptide chain through circular permutation could impact functional, soluble protein expression and interactions with cellular proteostasis factors. Here, we measure the solubility and function of every possible circular permutant (CP) of HaloTag in Escherichia coli cell lysate using a gel-based assay, and in living E. coli cells via FACS-seq. We find that 78% of HaloTag CPs retain protein function, though a subset of these proteins are also highly aggregation-prone. We examine the function of each CP in E. coli cells lacking the cotranslational chaperone trigger factor and the intracellular protease Lon and find no significant changes in function as a result of modifying the cellular proteostasis network. Finally, we biophysically characterize two topologically interesting CPs in vitro via circular dichroism and hydrogen-deuterium exchange coupled with mass spectrometry to reveal changes in global stability and folding kinetics with circular permutation. For CP33, we identify a change in the refolding intermediate as compared to wild-type (WT) HaloTag. Finally, we show that the strongest predictor of aggregation-prone expression in cells is the introduction of termini within the refolding intermediate. These results, in addition to our finding that termini insertion within the conformationally restrained core is most disruptive to protein function, indicate that successful folding of circular permutants may depend more on changes in folding pathway and termini insertion in flexible regions than on the availability of proteostasis factors.