Date of Award

Spring 5-24-2022

Document Type

Thesis (Ph.D.)

Department or Program

Chemistry

First Advisor

Dean Wilcox

Abstract

Metals are required for life. Many metalloproteins contain cysteine in their metal-binding site (MBS) and cysteines are unique in that they are reactive, and strongly bind certain metals, which aid in metal selectivity and specificity. Using isothermal titration calorimetry (ITC), the thermodynamic foundation for metal binding, cellular protection, and transcriptional regulation, which all utilize cysteines in their MBS, are quantified.

In bacteria there are metalloprotein pathways that actively uptake mercury, which are regulated by the metalloregulatory protein MerR. MerR de-represses the transcription of these mer proteins in a metal-dependent manner. Using ITC, the thermodynamic foundation of the negative allosteric coupling that regulates MerR is quantified. Within this regulated pathway is a metallochaperone in the periplasm of the cell, MerP, which functions as a mercury buffer, protecting the cell from the deleterious effects of the metal. The thermodynamic foundation of cellular protection and the mechanism of metal binding is characterized.

MerP is a part of a class of metallochaperones that all contain the same protein architecture and MBS. However, this class of metallochaperones binds a wide range of metals, including zinc, copper, and mercury. How one protein scaffold with the same MBS modulates metal binding is unknown. The fundamental thermodynamics suggest that second-sphere interactions are capable of modulating metal-binding properties of these ferredoxin-like folded proteins leading to metal selectivity and specificity.

Unlike bacterial mer protein, which generally bind one metal ion, other cysteine-rich proteins bind multiple metals simultaneously. In this work, the thermodynamics and mechanism of copper and zinc binding to human metallothionein (MT), a protein with many cellular functions in the regulation of metal toxicity and neuron growth inhibition, were quantified.

The binding of copper, zinc, and mercury to bacterial copper storage proteins (CSPs), which also utilize a large number of cysteine residues to bind and store copper, but unlike MT are conformationally stable. Metal binding in CSPs does not alter the global protein dynamics, thus a comparison between CSPs and MT provide valuable insight into how protein dynamics and the contribution of the protein scaffold can modulate the binding of different metals in solution.

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