Proof of principle in a de novo designed protein maquette: an allosterically regulated, charge-activated conformational switch in a tetra-alpha-helix bundle.

New understanding of the engineering and allosteric regulation of natural protein conformational switches (such as those that couple chemical and ionic signals, mechanical force, and electro/chemical free energy for biochemical activation, catalysis, and motion) can be derived from simple de novo designed synthetic protein models (maquettes). We demonstrate proof of principle of both reversible switch action and allosteric regulation in a tetra-alpha-helical bundle protein composed of two identical di-helical subunits containing heme coordinated at a specific position close to the disulfide loop region. Individual bundles assume one of two switch states related by large-scale mechanical changes: a syn-topology (helices of the different subunits parallel) or anti-topology (helices antiparallel). Both the spectral properties of a coproporphyrin probe appended to the loop region and the distance-dependent redox interaction between the hemes identify the topologies. Beginning from a syn-topology, introduction of ferric heme in each subunit (either binding or redox change) shifts the topological balance by 25-50-fold (1.9-2.3 kcal/mol) to an anti-dominance. Charge repulsion between the two internal cationic ferric hemes drives the syn- to anti-switch, as demonstrated in two ways. When fixed in the syn-topology, the second ferric heme binding is 25-80-fold (1.9-2.6 kcal/mol) weaker than the first, and adjacent heme redox potentials are split by 80 mV (1.85 kcal/mol), values that energetically match the shift in topological balance. Allosteric and cooperative regulation of the switch by ionic strength exploits the shielded charge interactions between the two hemes and the exposed, cooperative interactions between the coproporphyrin carboxylates.