Are protein folding intermediates the evolutionary consequence of functional constraints?

High-resolution experiments on several apparently two-state proteins point to the existence of partially structured excited- or intermediate-states in dynamic equilibrium with native states. Are these intermediate states the byproducts of functional constraints that are by necessity evolutionarily conserved or are they merely the hidden imprints of evolutionary processes? To investigate this, we characterize the folding of Barstar that has a rich history of complex conformational behavior employing a combination of methods-statistical-mechanical model, electrostatic calculations, MD simulations and multiple-sequence alignment-that provide a detailed yet consistent view of its landscape in agreement with experiments. We find that the multistate folding in Barstar is the direct consequence of a strong evolutionary pressure to maintain its binding affinity with Barnase through a large negative electrostatic potential on one face. A single mutation (E76K or E80K) at the binding site is shown to not only enhance the native-state stability but also alter the Barstar folding mechanism to resemble an unfrustrated two-state-like system. Our results argue that though natural proteins are expected to be minimally frustrated, functional constraints can singularly determine the folding mechanism even if it occurs at the expense of frustrated multistate folding.