This proposal describes a small-molecule fluorescence strategy to selectively label and thereby image discrete protein conformations and assemblies in live cells. We refer to this strategy as bipartite tetracysteine display. It is known that recombinant proteins containing the linear tetracysteine sequence Cys-Cys-Pro-Gly-Cys-Cys are selectively labeled by cell-permeable, pro-fluorescent biarsenical dyes FlAsH and ReAsH. Here we explore (Aim 1.1) whether the linear tetracysteine sequence can be split between two members of a protein partnership, or between two approximated regions of a single protein, while maintaining high biarsenical affinity and brightness, in vitro and in live cells.
Next (Aim 1. 2) we quantify the loss in FlAsH/ReAsH affinity and brightness when the protein or complex is misfolded or misassembled. Finally, (Aim 1.3) we describe a novel biotin-FlAsH conjugate designed to capture stable protein complexes or well-folded proteins from mixtures of less stable variants in live cells, allowing for the enrichment of phage or mammalian libraries for well-folded proteins or stable protein complexes.
In Aim 2 we build on the results of Aim 1 to develop encodable bipartite tetracysteine display-based sensors for (Aim 2.1) tyrosine kinase activity and (Aim 2.2) dynamic [Ca2+] changes that should be brighter than analogous FRET sensors and equipped with the added advantage of temporal control. Later experiments will develop analogous sensors for other kinases, histone methylation and phosphorylation. Finally, (Aim 2.3) we describe bipartite tetracysteine display-based sensors to identify molecules that rescue therapeutically relevant p53 mutants in live cells. Finally in Aim 3 we propose three applications of bipartite tetracysteine display that exploit the unique features of this technique.
Aim 3. 1 describes complex-edited FALI (CEF), in which FlAsH or ReAsH, upon irradiation, selectively inactivate not single proteins, as reported previously, but distinct protein-protein complexes.
Aim 3. 2 describes a related technique called complex-edited polymerization (CEB), in which ReAsH selectively polymerizes diaminobenzidine surrounding a distinct protein-protein complex. Finally in Aim 3.3 we design molecules to image and fluorescently differentiate alternative receptor tyrosine kinase (RTK) conformational states in the cell membrane. The application describes a small molecule fluorescence approach bipartite tetracysteine display to selectively label, image and/or inactivate discrete protein conformational states and assemblies in live cells. The proposed experiments apply bipartite tetracysteine display to develop brighter encodable sensors for protein kinases and dynamic changes in [Ca2+], for the detection of protein-protein interactions in vivo, and for the high-throughput screening of compounds that stabilize specific protein folds. It may also provide a means to study early protein misfolding events associated with Alzheimer's and Parkinson's disease and cystic fibrosis.
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