Project Summary With few exceptions, the enzymes that catalyze Nature?s most challenging and important reactions all require metal ions for activity. Among the most important of these metals in organisms from bacteria to humans are iron and manganese: nearly all organisms require iron for growth, and the few that do not require iron depend on manganese instead. Cells carefully regulate the concentrations of the metal ions that they require through a complex network of uptake proteins, export proteins, trafficking proteins, and protein- and RNA-based regulatory systems. The proper function of all of these systems are essential to avoid potentially deleterious chemistry of the ?free? metal ions as well as inactivation of enzymes resulting from incorporation of incorrect metal ions. As a result, mismanagement of the cellular free concentrations of iron and manganese has been connected with a number of human diseases, in particular neurodegeneration. On the other hand, the ability of a host organism (a human being) to disrupt metal management in an invading pathogen correlates with reduction of the virulence of that pathogen. Therefore, a more detailed understanding of the mechanisms of iron and manganese homeostasis in bacteria and humans has the potential to lead to new approaches to treat these diseases. An important approach to study metal homeostasis involves design and application of metal-selective fluorescent sensors ? consisting of either small molecules, proteins, or nucleic acids ? which are able to report on concentration, localization, and dynamics of the metal ions within cells. Fluorescent sensors for metals such as calcium and zinc have revolutionized our understanding of the biology of these ions. Unfortunately, few tools exist to study iron and manganese within cells, a reflection of the inherent challenges associated with selectively detecting an analyte that tends to bind weakly in biological systems. This research proposal outlines a comprehensive program to develop selective fluorescent sensors for iron and manganese, using Nature?s platforms for selective metal recognition, in order to probe novel mechanisms of metal regulation in bacteria and in eukaryotic cells. The proposal uses detailed biochemical analysis of metal recognition by iron- and manganese-binding proteins and nucleic acids to rationally design new sensors. These sensors are then deployed to provide insight into outstanding questions in the field, such as the mechanism and consequences of iron overload in pathogenic bacteria, as well as characterization of intracellular manganese trafficking systems in yeast and human cells. These answers will not only increase our understanding of fundamental mechanisms of selective metal recognition in biological systems but also potentially uncover new avenues for therapeutic intervention.
|Effective start/end date||9/5/20 → 8/31/21|