The Genetically Encoded Small Illuminant (GESI) technology proposed here consists of using an one-bead- one-compound (OBOC) combinatorial library screen to identify short peptide(s) that activate fluorophores/dyes, which can be coupled to alterations of their chemical environment including conformational change upon ligand binding and phosphorylation of the peptide. These peptides can then be genetically fused to a target protein to enable functional cellular imaging in vitro and in vivo. As proof of concept, we will fuse the identified peptide to integrin and use the exogenously added GESI-peptide-activating dye to demonstrate the utility of the proposed genetically encoded functional imaging system in living cells. We will test their ability to measure spatiotemporal dynamics, interactions with ligands and phosphorylation of intracellular domain. Integration of these distinct GESIs into one integrin protein will allow concurrent probing of multiple integrin functions in real time in living cell. Impact: GESI peptides can specifically bind to and activate the fluorescence of selected organic dyes. Some GESI peptides will do so only after binding to Ca2+, conformational changes or post-translationally modified, such as phosphorylation. Therefore, when transfected into a living cell, they can illuminate the spatiotemporal regulation and modification of proteins of interest. Like GFP, GESI can also be expressed in transgenic animal, or in tumor cells implanted into nude mice as xenograft. If the dye can be delivered to the tissue of interest, GESI reporting can occur in living animals as well. The genetic illuminants are small (1200- 1900 daltons), thus can be readily inserted along the sequence of the native proteins without interfering with their physiological functions. We can create functional peptide-dye pairs without overlapping spectrum to simultaneously monitor a number of PTMs or other cellular functions concurrently, to reveal system level regulation in living cells. This research will expand the catalogue of fluorescence imaging tools; their versatility will be broadly applicable to many fields of biology, including cancer.
Specific aims of this proposed project are:
Aim 1. Design and synthesis of a series of organic dyes suitable for GESI development.
Aim 2. Multiplex tracking of protein dynamics and ligand-receptor interactions with GESIs using integrin as a model system.
Aim 3. GESIs for post-translational modification of intracellular domain of integrin.
GESI: a novel technology for functional imaging in living cells Narrative: The proposed work builds on preliminary data that show the feasibility of designing, screening for, identifying, isolating, analyzing, and testing in vitro peptides that activate fluorescence of a single dye malachite green (MG). Here we expand on these studies by increasing the number of dyes to be screened against (including synthesizing new dyes; Aim 1) and developing and screening OBOC peptide libraries enable imaging of target protein localization and receptor- ligand interactions (Aim 2); and post translational modifications (Aim 3). As proof of concept, we will fuse the identified peptides/dye partners to integrin and test their ability to measure spatiotemporal dynamics, activation and phosphorylation. Integration of these distinct GESIs into one integrin protein will allow concurrent probing of multiple integrin functions in real time in living cell.
