Tuning the metal binding site specificity of a fluorescent sensor protein: from copper to zinc and back

Abstract

We previously reported the development of high affinity Zn²⁺ FRET sensors based on the Zn²⁺-mediated interaction between the CXXC motifs present in the copper chaperone proteins ATOX1 and WD4. By systematically substituting several of these cysteines for methionines, we constructed sensor variants that retain a high affinity for Cu⁺, while effectively abolishing their ability to form stable tetrahedral Zn²⁺ complexes.