Trustees of Indiana University
Grant: $47,210 - National Institutes of Health - May. 29, 2009
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Award Description: For the duration of the fellowship period, I propose a multidisciplinary approach utilizing protein chemistry techniques, thermodynamic methods, biological growth assays, and ratiometric zinc isotope analysis using ICP-MS to study metal homeostasis in S. pneumoniae. The specific aims of this proposal are: 1) To characterize the metal regulators AdcR and SczA. AdcR regulates the expression of a zinc uptake system in S. pneumoniae that is necessary for virulence and adhesion. I will elucidate using in vivo growth assays the important residues in AdcR for DNA and metal binding. SczA regulates the expression of the zinc efflux system, using standard fluorescence competition assays I will determine the affinity of SczA for zinc. 2) To evaluate zinc homeostasis in S. pneumoniae. Using isotope dilution and inductively couple mass spectrometry (ICP-MS), I will examine on a global level how deletions of proteins in the zinc homeostasispathway cause changes in global metal concentrations in the cell and the rate of zinc uptake as the cell reaches equilibrium. In addition, I will be able simultaneous examine how the zinc concentration effects mRNA levels and protein concentration. 3) To understand the nature of histidine loop in AdcA. By creating chimeric proteins of the manganese solute binding protein PsaA and the zinc solute binding protein AdcA, I will determine how the loop region of these proteins is able to discriminate between manganese and zinc using biophysical characterization, in vivo growth assays and ratiometric mass spectrometry.
Project Description: For the duration of the fellowship period, I propose a multidisciplinary approach utilizing protein chemistry techniques, thermodynamic methods, biological growth assays, and ratiometric zinc isotope analysis using ICP-MS to study metal homeostasis in S. pneumoniae. The specific aims of this proposal are: 1) To characterize the metal regulators AdcR and SczA. AdcR regulates the expression of a zinc uptake system in S. pneumoniae that is necessary for virulence and adhesion. I will elucidate using in vivo growth assays the important residues in AdcR for DNA and metal binding. SczA regulates the expression of the zinc efflux system, using standard fluorescence competition assays I will determine the affinity of SczA for zinc. 2) To evaluate zinc homeostasis in S. pneumoniae. Using isotope dilution and inductively couple mass spectrometry (ICP-MS), I will examine on a global level how deletions of proteins in the zinc homeostasispathway cause changes in global metal concentrations in the cell and the rate of zinc uptake as the cell reaches equilibrium. In addition, I will be able simultaneous examine how the zinc concentration effects mRNA levels and protein concentration. 3) To understand the nature of histidine loop in AdcA. By creating chimeric proteins of the manganese solute binding protein PsaA and the zinc solute binding protein AdcA, I will determine how the loop region of these proteins is able to discriminate between manganese and zinc using biophysical characterization, in vivo growth assays and ratiometric mass spectrometry.
Jobs Summary: Post Doctoral Appointee, Temp Post Doc Appointee (Total jobs reported: 1)
Project Status: Less Than 50% Completed
This award's data was last updated on May. 29, 2009. Help expand these official descriptions using the wiki below.
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