Proof of principle in a de novo designed protein maquette: An allosterically regulated, charge-activated conformational switch in a tetra-alpha-helixbundle

New understanding of the engineering and allosteric regulation of natural p rotein conformational switches (such as those that couple chemical and ioni c signals, mechanical force, and electro/chemical free energy for biochemic al activation, catalysis, and motion) can be derived from simple de novo de signed synthetic protein models (maquettes). We demonstrate proof of princi ple of both reversible switch action and allosteric regulation in a tetra-a lpha -helical bundle protein composed of two identical di-helical subunits containing heme coordinated at a specific position close to the disulfide l oop region. Individual bundles assume one of two switch states related by l arge-scale mechanical changes: a syn-topology (helices of the different sub units parallel) or anti-topology (helices antiparallel). Both the spectral properties of a coproporphyrin probe appended to the loop region and the di stance-dependent redox interaction between the hemes identify the topologie s. Beginning from a syn-topology, introduction of ferric heme in each subun it (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 demo nstrated in two ways. When fixed in the syn-topology, the second ferric hem e binding is 25-80-fold (1.9-2.6 kcal/mol) weaker than the first, and adjac ent heme redox potentials are split by 80 mV (1.85 kcal/mol), values that e nergetically match the shift in topological balance. Allosteric and coopera tive regulation of the switch by ionic strength exploits the shielded charg e interactions between the two hemes and the exposed, cooperative interacti ons between the coproporphyrin carboxylates.