Design and Development of Fluorescent Sensors for the Detection of Proximally Phosphorylated Peptide and Protein Motifs

While protein phosphorylation is recognized as a chief regulator of protein activity, little is known about the significance of the relative spatial separation among the phosphorylated sites. Specifically, while proximal phosphorylation on two or more residues appears to be associated with disease, no simple methods for the detection of this motif are commercially available. To address this, we developed a simple yet robust chemosensory approach, whose selectivity for proximally phosphorylated motifs relied upon the stoichiometry of sensor binding. A single chemosensor molecule, consisting of a pyrene excimer-forming fluorophore and a phosphate-binding Zn2+-cyclen unit, was predicted to associate with a single phosphorylated site. Thus, a proximally phosphorylated motif would be capable of binding more than one sensor molecule bringing their pyrene units into close proximity, which in turn would induce measurable excimer fluorescence. The turn-on mechanism of the developed approach, and its non-covalent mode of action, enabled the application of this system towards the detection of proximally phosphorylated peptides and proteins in a variety of widely used research platforms. These include aqueous solutions, polyacrylamide gels, blotting membranes, microplate surfaces, as well as in cell imaging. A powerful feature of this approach in gel and blot assays, was its compatibility with additional downstream analyses using other stains as well as mass spectrometry. The signal detection could be achieved using standard equipment available to a majority of laboratories. In the course of this work we have extensively optimized this sensory system from the biophysical and chemical perspectives. Guided by the classical principles of molecular recognition, this thesis work yielded the most efficient chemosensory system for the detection of proximally phosphorylated proteins described to date, and adds significant value to the fields of fundamental phospho-proteomics, disease characterization and drug development.Ph.D.2017-11-30 00:00:0