ANYL 138 |
| Calmodulin (CaM) is a calcium signaling protein that binds to wide variety of target proteins. Determining the binding affinities of CaM with target complexes is crucial for understanding the nature of interaction under various conditions such as altered targets. Binding affinities have typically been measured indirectly, eg. from kinetic measurements. In this work we have developed a rapid method for measuring Kd of CaM target complexes based on the fluorescence polarization (FP). FP assays are rapid, sensitive homogeneous assays suitable for high throughput screening formats. We explored the feasibility of this assay using a CaM target protein, plasma membrane calcium ATPase (PMCA). PMCA activity is regulated by binding of CaM. We measured the changes in anisotropy of CaM labeled with Oregon green 488 upon titration with PMCA. Then we derived a simple binding model to fit the experimental data to obtain the Kd. This FP assay gave a direct measurement of the Kd of CaM with PMCA. The value we derived from this assay (7 nM ± 2 nM) is consistent with the previous measurements. We also investigated the Calcium ion dependence of CaM binding to PMCA. The Calcium dependence follows a simple Hill plot demonstrating cooperative binding of Calcium ions to the binding sites in CaM. |
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General Posters
7:00 PM-9:00 PM, Sunday, August 19, 2007 BCEC -- Exhibit Hall - B2, Poster
Division of Analytical Chemistry |