Protein Selectivity Factors as a Molecular Basis for Metal Toxicity

Abstract

Toxic metals are known to displace physiologically-relevant metal ions in proteins, and may activate or deactivate protein function in response to changes in the free metal concentration. To investigate potential relationships between metal/protein complexes and toxicity, an analysis of proteins structural and sequential data was used to establish statistical bases for identifying key selectivity factors associated with Pb2+-protein binding. These data led us to hypothesize that Pb2+, and potentially other toxic metals, may induce opportunistic binding in regions of negative electrostatic potential, thus altering the proteins conformation.To compare structural/conformational changes, investigate selectivity and affinity, and probe the mechanism of toxic metal-protein interactions, several natural and engineered Ca2+-binding proteins (CaBPs) were analyzed using Fluorescence, CD and 1D and 2D NMR spectroscopy. Engineered proteins were developed based on grafting methods that involved insertion of metal-binding motifs in flexible regions of protein scaffolds to investigate biophysical properties associated with binding reactions in isolated sites. Additionally, the ubiquitous signaling protein calmodulin (CaM) was evaluated extensively to determine changes associated with competitive binding between Ca2+ and anthropogenically available toxicants such as Pb2+, Gd3+, La3+, Tb3+ and In3+. Results suggest that certain toxic metals may not only displace the biologically-relevant metals in metalloproteins, but support our hypothesis that opportunistic binding occurs in non-sites. This has important implications for the potential binding of toxic metals by non-metalloproteins, as well as providing a basis for understanding the impact of toxicity related to downstream protein-protein interactions.