Paralogous proteins play a vital role in evolutionary adaptation of organisms and species divergence. One outstanding question is the molecular basis for how folding mechanisms differ in paralogs that not only exhibit similar topologies but also evolve under near-identical selection pressures. Here, we address this question by studying a paralogous protein pair from enterobacteria, Hha and Cnu, combining experiments, simulations and statistical modeling. We find that Hha is less stable and folds an order of magnitude slower than Cnu despite similar packing and topological features. Differences in surface charge–charge interactions, however, promote a N-terminal biased unfolding mechanism in Hha unlike Cnu that unfolds via the C terminus. Our work highlights how electrostatic frustration contributes to the population of heterogeneous native ensembles in paralogs and the avenues through which evolutionary topological constraints could be overcome by modulating charge–charge interactions.
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