Defining a Molecular Mechanism for Lead Toxicity via Calcium-Binding Proteins

Author

Michael KirbergerFollow

Date of Award

Spring 5-7-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Jenny J. Yang

Second Advisor

Markus Germann

Third Advisor

Aimin Liu

Fourth Advisor

Robert Wolhueter

Abstract

Essential metals like Ca²⁺ and Zn²⁺ play critical roles in biological processes through protein interactions. Conversely, non-essential metals (e.g., Gd³⁺ and Pb²⁺) also interact with proteins, often with toxic effects. Molecular metal toxicity is assumed to be due to ionic displacement, and studies have demonstrated that Pb²⁺ replaces Zn²⁺, Ca²⁺ and other essential metals in proteins. The focus of this work was to compare protein Ca²⁺ and Pb²⁺ -binding sites and to investigate a mechanism of Pb²⁺ toxicity in Ca²⁺-binding proteins, particularly the intracellular trigger protein calmodulin (CaM) which binds four Ca²⁺ ions and interacts with numerous molecular targets via Ca²⁺-induced conformational change.

A statistical analysis of PDB structural data for Pb²⁺ and Ca²⁺-binding (EF-hand and non-EF-hand) proteins revealed fewer binding ligands in Pb²⁺ sites (4 ± 2), than non-EF-Hand (6 ± 2) and EF-Hand (7 ± 1) Ca²⁺-binding sites. Pb²⁺ binds predominantly with sidechain Glu (38.4%), which is less prevalent in both non-EF-Hand (10.4%) and EF-Hand (26.6%) sites. Interestingly, analyses of proteins where Pb²⁺ replaces Ca²⁺ (calmodulin) or Zn²⁺ (5-aminolaevulinic acid dehydratase) revealed structural changes presumably unrelated to ionic displacement. These results suggested that Pb²⁺ adopts diverse binding geometries and that opportunistic binding outside of known Ca²⁺-binding sites may play a role in molecular metal toxicity.

Ca²⁺-binding affinities (K_d) using phenylalanine and tyrosine fluorescence were found to be 1.15 ± 0.68 X 10^-5 M and 2.04 ± 0.02 X 10^-6 M for the N- and C-terminal domains, respectively. The K_d for Pb²⁺-binding in the N-terminal domain, 1.40 ± 0.30 X 10^-6 M, was 8-fold higher than Ca²⁺. Binding of Pb²⁺ in the C-terminal domain produced a biphasic response with K_d values 7.34 ± 0.95 X 10^-7 M and 1.93 ± 0.32 X 10^-6 M, suggesting a single higher affinity Pb²⁺-binding site in the C-terminal domain with nearly equivalent affinity for the remaining sites. Competitive effects of Pb²⁺ added to Ca²⁺-loaded CaM were examined using multiple NMR techniques. Pb²⁺ was found to displace Ca²⁺ only in the N-terminal domain, however structural/dynamic changes were observed in the central helix apparently due to Pb²⁺-binding in secondary sites. These data supported our hypothesis that CaM structure and function is altered by opportunistic Pb²⁺-binding.

DOI

https://doi.org/10.57709/2001394

Recommended Citation

Kirberger, Michael, "Defining a Molecular Mechanism for Lead Toxicity via Calcium-Binding Proteins." Dissertation, Georgia State University, 2011.
doi: https://doi.org/10.57709/2001394