Model protein surface domains for the investigation of metal ion-dependent macromolecular interactions and biospecific metal ion transfer

Abstract

We describe the use of surface-immobilized transition metal ions not only as tools for the site-specific immobilization and purification of peptides and proteins but also as probes of altered macromolecular surface architecture. Although we present examples of the use of traditional immobilized chemical chelators (e.g., iminodiacetate) for these purposes, we also introduce methods for identifying and using natural metal-binding domains derived from protein surfaces to immobilize metal ions in a biospecific manner. Such model protein surfaces facilitate the investigation of the mechanisms by which metal ions influence the interaction of macromolecules. This approach, immobilized metal-binding domain recognition, depends on the proficient identification and characterization of individual protein surface metal-binding domains. To facilitate this process, we have developed the use of two different forms of soft ionization mass spectrometry to observe intact peptide-metal ion complexes; this enables the mass-dependent identification of specific metal-binding sequences. Model protein surfaces constructed thus far and found to exhibit metal ion-dependent macromolecular recognition specificity include (i) a series of immobilized peptides (GHHPH) n G, which define the surface metal-binding domain (G365-H389) of the human plasma metal ion transport protein known as histidine-rich glycoprotein; (ii) the immobilized dimerization domain (D473-L525) from the human estrogen receptor protein; (iii) the immobilized “zinc-finger” DNA-binding domain (K180-M250) of the human estrogen receptor; and (iv) the immobilized iron-binding domain (R1-K18) derived from the N-terminus of human β-casein. We discuss the investigation of metal ion-dependent interactions between macromolecules and the transfer of metal ions between macromolecules.