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

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.