RESUMEN
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.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Pliegue de Proteína , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Solubilidad , CinéticaRESUMEN
The evolutionary origins of complex morphological structures such as the vertebrate eye or insect wing remain one of the greatest mysteries of biology. Recent comparative studies of gene expression imply that new structures are not built from scratch, but rather form by co-opting preexisting gene networks. A key prediction of this model is that upstream factors within the network will activate their preexisting targets (i.e., enhancers) to form novel anatomies. Here, we show how a recently derived morphological novelty present in the genitalia of D. melanogaster employs an ancestral Hox-regulated network deployed in the embryo to generate the larval posterior spiracle. We demonstrate how transcriptional enhancers and constituent transcription factor binding sites are used in both ancestral and novel contexts. These results illustrate network co-option at the level of individual connections between regulatory genes and highlight how morphological novelty may originate through the co-option of networks controlling seemingly unrelated structures.