A Search for Zn(II) Metallochaperones in E. coli, Proteomic and Genomic Approaches
Zinc is a transition metal that exists exclusively in the +2 oxidation state in biological systems (Zn(II)). Zn(II) serves as a catalytic cofactor in members from all classes of enzymes and as a structural component in dozens of proteins. Despite being essential for the structural/catalytic properties of many cellular proteins, excess Zn(II) is toxic to cells. Recent reports demonstrate that E. coli cells contain low millimolar concentrations of Zn(II); however, subsequent studies reveal that there are no pools of free Zn(II) in the cytoplasm of the cell. Several groups have hypothesized the existence of Zn(II)-metallochaperones, which bind and deliver Zn(II) ions; however, no such proteins have yet been identified. This dissertation describes our efforts to identify the Zn(II)-responsive proteins in E. coli by using proteomics (2D gels coupled with peptide identification) and genomics (cDNA microarrays). Proteomic studies identified a number of proteins with differential expression levels in response to extracellular Zn(II) levels; however, none of the identified proteins have previously been associated with Zn(II) transport. cDNA microarrays identified a number of differentially-expressed transcripts in response to stress by Zn(II) excess and deficiency, and several of the corresponding proteins were hypothesized to be Zn(II)-metallochaperones. One candidate protein, YodA, was cloned, over-expressed, and used as bait in a pulldown assay. Comparisons of results from the proteomic and genomic approaches demonstrate a surprising lack of consistency, which indicates that caution should be used when applying these techniques to study global protein/RNA changes in response to external stimuli. In summary, this dissertation describes an approach to probe for the Zn(II) metallome of an organism, and this approach can be extended to other organisms to better understand the homeostatic pathways used to maintain intracellular metal ion concentrations.
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:e coli zinc transport and homeostasis proteomics cdna microarray 2d gel metal
Date of Publication:01/01/2005