SWTY--a general peptide probe for homogeneous solution binding assay of 14-3-3 proteins |
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Authors: | Wu Meng Coblitz Brian Shikano Sojin Long Shunyou Cockrell Lisa M Fu Haian Li Min |
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Affiliation: | Department of Neuroscience and High Throughput Biology Center, School of Medicine, Johns Hopkins University, 733 North Broadway, Baltimore, MD 21205, USA. |
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Abstract: | Dimeric 14-3-3 proteins exert diverse functions in eukaryotes by binding to specific phosphorylated sites on diverse target proteins. Critical to the physiological function of 14-3-3 proteins is the wide range of binding affinity to different ligands. The existing information of binding affinity is mainly derived from nonhomogeneous-based methods such as surface plasmon resonance and quantitative affinity precipitation. We have developed a fluorescence anisotropy peptide probe using a genetically isolated 14-3-3-binding SWTY motif. The synthetic 5-(and-6)-carboxyfluorescein(FAM)-RGRSWpTY-COOH peptide, when bound to 14-3-3 proteins, exhibits a seven-fold increase in fluorescence anisotropy. Different from the existing assays for 14-3-3 binding, this homogeneous assay tests the interaction directly in solution. Hence it permits more accurate determination of the dissociation constants of 14-3-3 binding molecules. Protocols for a simple mix-and-read format have been developed to evaluate 14-3-3 protein interactions using either purified recombinant 14-3-3 fusion proteins or native 14-3-3s in crude cell lysate. Optimal assay conditions for high-throughput screening for modulators of 14-3-3 binding have been determined. |
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Keywords: | 14-3-3 Fluorescence anisotropy Binding assay High-throughput screening Dissociation constant SWTY |
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