| Although the toxicity of lead has been recognized for centuries, the molecular mechanism of lead toxicity remains a mystery. Recent studies suggest that lead is able to displace zinc from structural zinc-binding sites that are rich in cysteine residues. One such protein is the glucocorticoid receptor DNA-binding domain (GR-DBD), which plays an integral role in regulating many developmental pathways. Hence, the purpose of this thesis is to describe the interactions of zinc and lead with the GR-DBD protein as a possible mechanism of lead toxicity.;The binding affinity of zinc to the GR-DBD was determined through the use of metal-binding competition experiments in which Co2+ was used as a spectroscopic probe. In this manner, the dissociation constants of Co2+ from the GR-DBD were calculated: K d1Co = 4.1 (±0.6) × 10−7 M; Kd2Co = 1.7 (±0.3) × 10−7 M. Further studies indicated that Zn2+ binds to each of the Cys4 metal-binding sites approximately three orders of magnitude more tightly than Co2+ does: K d1Zn = 2 (±1) × 10 −10 M; Kd2Zn = 3 (±1) × 10−10 M for the GR-DBD. Similarly, the interactions of Pb2+ with the GR-DBD were studied by performing a series of metal-biding titrations. These studies indicate that Pb 2+ binds to the GR-DBD with roughly the same affinity as Zn 2+ (KdPb ∼ KdZn ∼ 10−10 M). Furthermore, Pb2+ was able to displace Zn2+ from the GR-DBD under physiologically relevant conditions. However, more than two molar equivalents of Pb2+ bind to the GR-DBD despite the fact that there are only two zinc-binding sites present in this domain.;One possible explanation for why more than two molar equivalents of Pb 2+ bind to the GR-DBD is the presence of two cysteine residues (Cys 500 and Cys450) that do not participate in zinc binding but are close to the zinc-binding sites. Metal-binding studies using three GR-DBD mutants revealed that the Cys500 and Cys450 residues participate in the binding of Pb2+, to the GR-DBD. Taken together, the results presented herein suggest that lead interactions with members of the nuclear hormone receptor superfamily may explain, in part, the toxicity of lead. |
