The goal of this project is to develop a novel class of Ca(ll) sensor proteins that will have wide applicability in studies of human diseases including various cardiomyopathies, Alzheimer diseases, cancer, and lens cataract formation that are known to be associated with altered Ca(ll) signaling. A major barrier to the understanding of specific spatio-temporal patterns of intracellular Ca(ll) signaling is the lack of targeted sensors that monitor concentration from submicromolar to greater than 5 mM. We have developed a novel approach for developing Ca(ll) sensors by grafting Ca(ll) binding motifs into Green Fluorescence Protein (GFP) that exhibit Ca(ll) dependent fluorescence changes. In addition, we can design de novo Ca(ll) binding sites in GFP such that they exhibit different Ca(ll) binding affinities and strong selectivity. This proposal has two objectives. First, we will develop GFP-based Ca(ll) sensors for the ER with a broad-range of Ca(ll) affinities (10 micromolar < Kd < 5mM) and optimized optical properties by grafting different Ca(ll) binding motifs. We will examine their optical properties, metal binding affinity and selectivity, stability and kinetic properties. The cellular locations of ER-directed Ca(ll) sensors will be verified cytochemically and immunocytochemically. We will monitor changes in ER Ca(ll) signaling in real time in response to agonists such as ATP and histamine. Second, we will develop high affinity and selective Ca(ll) sensors for the cytosol (0.1 <Kd < 10 microM) by designing de novo Ca(ll) binding sites within GFP. We will enhance metal binding affinities and selectivity of Ca(ll) sensors with different types of Ca(il) ligand residues and number of charged residues. In addition to optimizing optical properties and kinetic properties of the Ca(ll) sensors, we will evaluate our developed Ca(ll) sensors in vivo by varying the intracellular cytosolic Ca(ll) concentration using Ca(ll) ionophores and appropriate Ca(ll) pump inhibitors, and comparing the estimated Ca(ll) concentrations with that derived using small synthetic dyes and currently available CaM-based sensors. Calibration method for in vivo detection of [Ca(ll)] will be developed using established HeLa cell lines and primary lens cell cultures. The proposed work will have a significant impact on the field because it will not only significantly expand the repertoire of tools for studying cell signaling and related diseases, but also it will result in technological advances toward designing Ca(ll) switches that are capable of controlling protein function. [unreadable] [unreadable]