Multivalent protein binding to metal-complexing materials : applications to synthetic receptors and affinity chromatography

Citation

Johnson, Robert David (1995) Multivalent protein binding to metal-complexing materials : applications to synthetic receptors and affinity chromatography. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/6kdk-t643. https://resolver.caltech.edu/CaltechETD:etd-10122007-091509

Abstract

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This investigation demonstrates that proteins have the capability to bind simultaneously to multiple transition metals of metal-complexing materials. This finding has important implications for the design of novel materials for protein recognition. Our approach to protein recognition, based on intermolecular metal-to-ligand interactions, matches a protein's unique pattern of histidines with a complementary arrangement of transition metal complexes.

A model system is used to demonstrate the validity of this approach in the simplest case by matching the distance between metal ions of rationally designed bis-metal [...] "receptors" to that between imidazoles of bis-imidazole "targets." This system additionally demonstrates how other features of receptor design can influence binding selectivity. A 2D NMR procedure is developed to measure directly protein surface histidine binding to copper complexes, and subsequently demonstrates that the local environment of the histidine and the structure of the copper complex can modulate individual copper-histidine interactions. Thus it may indeed be possible to design metal-containing receptors which are able to form simultaneous metal-ligand bonds with a specific arrangement of protein metal-coordinating groups.

There are two important obstacles preventing a similarly detailed description of protein binding to metal-complexing surfaces: protein adsorption may involve binding to one or more metal sites, and a detailed description of the geometry of surface metal sites would be hopelessly complex. We can, however, apply the microscopic concept of simultaneous metal-ligand interactions to interpret the macroscopic phenomena of protein partitioning in immobilized metal affinity chromatography (IMAC). In this context, the ability of commercially available IMAC materials to support multiple protein-surface interactions is shown to be dependent on three factors: the number of histidines on the protein (as manipulated by site directed mutagenesis), the number of deprotonated amino groups on the protein (pH control), and the density of binding sites on the surface (copper loading). These results demonstrate that a realistic description of protein binding in IMAC must consider a heterogeneous population of surface binding sites. In IMAC this is shown to be conveniently expressed by the Temkin isotherm, making it an instructive model to explore heterogeneity displayed by other chromatographic materials and by biological systems.

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