Principles and practice of determining metal-protein affinities.

Tessa R Young,Zhiguang Xiao

doi:10.1042/bcj20200838

Abstract

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

Highlights

Metal–protein interactions have played many critical roles in biology since life first evolved
We review a selection of spectroscopic probes which can be used to determine protein affinities for essential biological transition metals (including Mn(II), Fe(II), Co(II), Ni(II), Cu(I), Cu(II) and Zn(II)) and, using selected examples, demonstrate how rational probe selection combined with prudent experimental design can be applied to determine accurate KD values
Innovative new research has quantified the thermodynamic availability of common transition metals inside a cell [4,5], making it possible to predict in vivo metal occupancy of proteins using their in vitro affinity data as input [6]

Summary

Introduction

Metal–protein interactions have played many critical roles in biology since life first evolved. The apparent affinity estimated by direct metal titration (with the experimental data fitted to equation 5) was aKD (P) = 10−8.0 M which agreed with the conditional KD(P) = 10−8.1 M determined by competition with the standard ligand glycine [19].

Results

Conclusion