Maintaining proper cellular levels of metal ions is key to the survival of all organisms. The viability of bacteria, including human pathogens, has been linked to the ability to acquire or compete for transition metals. Several human diseases have been shown to result from a breakdown in cellular metal trafficking. In the case of transition metals, maintaining proper metal homeostasis involves controlling a delicate balance between optimal levels required to have functional enzymes, etc., and the concentration at which the metals become toxic. To achieve this control, organisms have developed mechanisms to ensure the acquisition of specific metals necessary for growth, exclude toxic metals, and control their intercellular levels. These metal trafficking systems rely on proteins that have the ability to distinguish between metal ions that often have similar sizes and charges. The metalloproteins involved, collectively known as metal trafficking proteins, control uptake and efflux (metallotransporters), target the delivery of metals to specific enzymes (metallochaperones) and regulate the expression of the other proteins in response to metal ion concentration (metalloregulators), etc. Although many examples of proteins that achieve these functions for various metal ions have been characterized, the mechanisms that allow for specific metal recognition and metal-specific biological responses are not well known. The overall objective of the proposed research is to understand the structural parameters that that allow trafficking proteins to distinguish between metals, and the related protein structural changes that drive metal specific biological responses. Toward this goal, we plan to examine structural parameters that are involved in metal-recognition by a metallochaperone (HypA) and a metalloregulator (RcnR) in Helicobacter pylori and E. coli, respectively. The approach involves cloning and expressing the proteins, characterizing their metal ion affinities, elucidating the structure of th metal sites and proteins, and assessing protein-protein interactions. To do this, mutagenesis and several structural techniques including X-ray absorption spectroscopy (XAS), crystallography, NMR, and mass-spectrometry-based techniques, are utilized. To assess function in this diverse group of proteins, assays specific to each protein will be employed; a transcription reporter assay for RcnR, and assays that address the ability of the protein to deliver metals to the target enzymes for the metallochaperone. In addition to the understanding of the basic biochemistry, a detailed understanding of the molecular mechanisms involved in metal trafficking may lead to the development of therapies for the treatment of patients with metal overloads (including poisoning) or deficiencies/excesses resulting from defects in metal metabolism, and to the design of new antibiotics that interfere with bacterial metal metabolism.
The research proposed seeks to provide a detailed understanding of the molecular mechanisms involved in cellular metal trafficking. This information will be useful in the development of therapies for the treatment of patients with metal overloads (including poisoning) or deficiencies resulting from defects in metal metabolism, and in the design of new antibiotics that interfere with bacterial metal metabolism.
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