In this proposal, Dr. Ruthstein and her new group at the chemistry department in Bar-Ilan University, Israel, will determine the sensing and transcription mechanisms of metal ion sensors in bacteria. Metals are commonly found as natural constituents of proteins; however, many metal ions can be toxic when free in biological fluids. Hence, microorganisms have evolved considerable regulatory machinery to control the intracellular and extracellular concentrations and types of metal ions encountered in their microenvironments. Gene regulatory proteins, called metal sensor proteins mediate this metal traffic control. These proteins have evolved metal coordination sites that “sense” specific metal ion and activate or inhibit DNA binding or transcription activation, thereby controlling the expression of genes that mediate what must be an exquisitely selective adaptive response. In order to understand how the sensor senses a specific metal and by this initiates a biological process, one has to be able to probe strutuctural changes that occur in the protein as a function of binding of the correct and wrong metal ion, and the binding of the DNA. This work will use site-directed spin labeling together with pulsed electron paramagnetic resonance spectroscopy (EPR) to shed light on such processes. EPR is an excellent tool to resolve systems that evolve protein-protein interaction and protein-DNA interaction. Distances up to 80 Å between two paramagnetic spin labels can be measured by pulsed EPR. In some cases, the measurement of one distance (or a few) is sufficient to establish the plausibility of a mechanism or corroborate a proposed structure. This work will initially focus on two Cu+ sensors (CsoR, CueR) from CsoR and MerR families. The first family inhibits DNA binding upon metal binding and the second activates transcription, but the research will extend in the future to other metal sensors from each of the families.