Principles and practice of determining metal–protein affinities

Metal ions play many critical roles in biology, as structural and catalytic cofactors, cell regulatory signalling elements. The metal–protein affinity, expressed conveniently by the metal dissociation constant, KD, describes thermodynamic strength of a interaction is key parameter that can be used, for example, to understand how proteins may acquire metals identify dynamic elements (e.g. cofactor binding, changing availabilities) which regulate protein metalation vivo. Here, we outline fundamental principles practical considerations are reliable quantification affinities. We review selection spectroscopic probes used determine affinities essential biological transition (including Mn(II), Fe(II), Co(II), Ni(II), Cu(I), Cu(II) Zn(II)) and, using selected examples, demonstrate rational probe combined with prudent experimental design applied accurate KD values.