A Ca2+-Sensing Molecular Switch Based on Alternate Frame Protein Folding

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Abstract
Existing strategies for creating biosensors mainly rely on large conformational changes to transduce a binding event to an output signal. Most molecules, however, do not exhibit large-scale structural changes upon substrate binding. Here, we present a general approach (alternate frame folding, or AFF) for engineering allosteric control into ligand binding proteins. AFF can in principle be applied to any protein to establish a binding-induced conformational change, even if none exists in the natural molecule. The AFF design duplicates a portion of the amino acid sequence, creating an additional “frame” of folding. One frame corresponds to the wild-type sequence, and folding produces the normal structure. Folding in the second frame yields a circularly permuted protein. Because the two native structures compete for a shared sequence, they fold in a mutually exclusive fashion. Binding energy is used to drive the conformational change from one fold to the other. We demonstrate the approach by converting the protein calbindin D9k into a molecular switch that senses Ca2+. The structures of Ca2+-free and Ca2+-bound calbindin are nearly identical. Nevertheless, the AFF mechanism engineers a robust conformational change that we detect using two covalently attached fluorescent groups. Biological fluorophores can also be employed to create a genetically encoded sensor. AFF should be broadly applicable to create sensors for a variety of small molecules.