Site-Specific Protein Labeling using Reversible Transition Metal Ion Binding

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Fluorescence spectroscopy is a powerful tool for studying the structure and conformational dynamics of protein molecules both in isolation and in their cellular context. Fluorescence experiments frequently employ small, cysteine-reactive fluorophores. However, it can be difficult to obtain specific labeling of a desired cysteine in proteins with multiple cysteines or in a protein's native environment in which many cysteine-containing proteins are present. To obtain specific labeling, we have developed a method where a desired cysteine can be reversibly protected by binding transition metal ions (e.g. Cd2+ and Zn2+) while background cysteines are blocked with non-fluorescent covalent modifiers. Following removal of transition metal ions, the deprotected cysteine is then available to specifically react with a fluorophore. In order to protect specific cysteines, the affinity for metal binding was increased by placing metal binding residues nearby in regions of known secondary structure (e.g. an alpha-helix) to act as coordination partners in a metal binding site. The placement of histidine residues next to or one turn away from a cysteine in an alpha-helix increased the metal binding affinity of that cysteine. This allows for protection of a particular cysteine at much lower metal concentrations. Using pairs of cysteines, rather than a combination of cysteines and histidines, further stabilized metal binding. Finally, we demonstrate the ability of this technique to selectively label a particular cysteine in mixtures of proteins containing cysteines. These motifs are simple, can be introduced into proteins with minimal perturbation, and allow for selective labeling of sites within a protein, specific proteins in a mixture, or proteins in a native environment. This technique has great potential for use in patch-clamp fluorometry studies of ion channels in which non-specific labeling of patch-associated proteins makes it difficult to resolve fluorescence from channel proteins.