Spatial confinement of macromolecules within specialized cellular compartments is a key strategy of living systems to control <i>where</i> and <i>when</i> chemical reactions may take place. The role of boundaries however goes beyond the simple physical separation between reactants and products, eventually affecting the entropy of the confined macromolecule. Recent studies clearly demonstrate that proteins are thermodynamically stabilized in small volumes, due to the limited probability of extended unfolded configurations and that the binding of multiple ligands constrained in the vicinity of their targets is greatly favored by a synergistic effect known as “entropic avidity”. In the past few years, several DNA-based nanostructures have been realized for the immobilization of single proteins and enzyme cascades, resulting in the consistent observation that proteins behave differently when anchored to a DNA surface; however, the general mechanisms behind this phenomenon are still partially unknown. In this work, we modified a previously reported DNA origami frame for the selective trapping of one single thrombin molecule using non-covalent aptamer ligands anchored to the inner sides of the cavity (Fig. 1). We showed that the number, type and intermolecular distance between the thrombin-binding aptamers, as well as their relative spatial orientation within the DNA frame, greatly affect both the avidity and proteolytic activity of the enzyme and propose a model, based on molecular dynamics simulations, to rationalize the observed data. Our results demonstrate that both design-independent electrostatic effects and design-dependent allosteric factors regulate the binding-to-activity coupling of the DNA-tethered protein, thus providing a tool to control protein’s reactivity through its spatial confinement within customized DNA boundaries.

DNA-boundaries enhance the binding affinity and proteolytic activity of thrombin

256th National Meeting (2018)

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