University of North Carolina at Chapel Hill Graduate School
Doi
Abstract
Synthetic peptide reporters are useful biochemical tools in the field of biomedicalresearch with diverse applications ranging from use as substrates for recombinant enzymes, celllysates, bulk live cells, and even single mammalian cells. The benefits of utilizing peptidesinclude their relatively straightforward synthesis, highly customizable nature, high shelf-life, andfacile derivatization with fluorescent tags to facilitate fluorescent detection. Additionally,peptides can be engineered to display a wide range of non-native modifications that increasetheir resistance to proteolytic degradation and mitigate post-translational modifications at keysites to temporally control reactions with target enzymes. Particularly, photoactivatableprotecting groups have become an attractive option for masking phosphorylation sites onpeptides in the presence of recombinant enzymes and/or in live cells. Such photoactivatablepeptides display resistance to an array of enzymatic modifications until the application of aphotostimulus to unmask the protected sites on the peptide. Such innovations show promise forperforming temporally controlled enzymatic reactions within populations of live single cells.The work described in this dissertation covers the development of a small library of dualsubstrate peptide reporters for quantifying epidermal growth factor receptor kinase (EGFR) andprotein kinase B (Akt) activity in single cells. A suite of characterization experiments wereperformed to asses the relative affinities of each reporter for the target kinases, stability towardsproteolysis and dephosphorylation in cell lysates and bulk live cells, and selectivity for the targetkinases. From the results of these studies, a photoactivatable variant of the best candidate ivreporter was developed which employed photoactivatable 4,5-dimethoxy-2-nitrobenzylprotecting groups on the EGFR and Akt phosphorylation sites. The ability of this reporter toundergo photoactivation, resist phosphorylation and proteolytic degradation prior to theapplication of a photostimulus at 360 nm was assessed in the presence of recombinant enzymes,in bulk live cells, and at the single cell level. Additionally, while the photoactivatable reporterwas developed to control the initiation of enzymatic reporter modifications in cells, aphotoactivatable chemical fixative was developed in an attempt to control the halting ofintracellular reactions and facilitate the storage of reporter loaded cells until later analysis. Thegoal of having a photoactivatable fixative was to enable the protective encapsulation of fixedcells in a hydrogel with the goal of preserving cellular membrane integrity of fixed reporterloaded cells until a later time at which photolysis of the hydrogel would facilitate analysis ofsinge reporter loaded cells. The reporter and fixative design and characterization strategiespresented in this dissertation are promising for the development of fully temporally controlledmeasurements of enzyme activity in single cells. Such innovations are expected to improve therigor of peptide reporter-based single cell experiments and facilitate better comparisons inenzyme activity profiles between ultra-small populations of single cells.Doctor of Philosoph
