Fluorescence techniques provide extraordinary high levels of sensitivity, specificity and selectivity and they are well-suited for high-throughput screening and imaging of specific proteins and drugs in biological samples. The most effective approach to quantify protein complexes within a sample is to use fluorescence anisotropy (FA) to quantify the formation of complexes between a FA-sensor and its target. FA has the distinct advantage over FRET in that it simply measures the increase in molecular volume of the bound FA sensor and using a single fluorophore. Currently, most FA-probes are prepared via laborious and specific chemical synthesis, reducing their appeal for high-throughput and in vivo screening of specific drugs or proteins. We will advance FA-based analyses of specific proteins for in vitro and in vivo systems through the introduction and optimization of a completely new class of genetically-encoded FA-sensor. The three sensors detailed in this proposal are truncated forms of: (i), a non-switchable mutant of Lov2 (fLov2), (ii), the yellow fluorescent protein from Vibrio fischeri (Y1);(iii), the lumazine binding protein (LUMP) from Photobacterium Leioghnati. The fluorescence properties of these flavoproteins proteins are similar to GFP, YFP and CFP respectively, although they only have 40%~67% of the mass, and exhibit far longer fluorescence lifetimes than GFP, a key property in the design of an FA-sensor for large proteins. The studies detailed are innovative on several counts and include the introduction of the smallest genetically-encoded fluorescent proteins for intracellular imaging of fused proteins, and the first encoded probes specifically designed for FA-based detection and imaging of specific protein targets in living cells. The research also identifies a promising approach for FA-based proteome-wide analysis of protein or drug interactions in vitro and in living bacteria and yeast.
The proposal will result in the introduction of a completely new class of genetically-encoded probe for sensitive, fluorescence anisotropy based detection and imaging of specific proteins or drugs within biological samples and in living cells. These studies will empower new quantitative approaches for rapid, high throughput, high-content and proteome-wide screening and quantification of specific protein interactions with applications in medical diagnostics, drug discovery and systems biology.
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