The detection of cellular events is central to understanding biomoleculer processes as well as aid in therapeutic intervention strategies. One of the most fascinating biomoleculer events during the life cycle of a cell is proteolytic cleavage of proteins by enzymes known as proteases. Proteases are ubiquitous and participate in essential functions such as fertilization, embryo development, cell cycle regulation, immune response, tissue remodeling and programmed cell death. As proteases are involved in fundamental cellular processes any dysregulation of protease activity is usually associated with a diseased state. Thus methods for detection of protease activity are desirable as it may facilitate the identification of many pathological conditions which are associated with the aberrant expression and activity of proteases.Towards the goal of a general and modular strategy we have utilized split protein reassembly and coiled coils to develop genetically encoded sensors for detection of proteases. We established our first generation protease design utilizing split firefly luciferase and anti-parallel coiled coils and detected Tobacco Etch Virus (TEV) as a model protease. Two further iterations of the coiled-coil design led to the development of second and third generation of protease sensors which showed substantial improvement in the sensor response and was applied towards detection of therapeutically relevant proteases such as caspase-3, prostate specific antigen (PSA), ß-secretase and calpain-1.We applied our methodolgy to develop protease biosensors for the detection of a family of cysteine protease known as caspases. Caspases are involved in programmed cell death and their misregulation is implicated in cancer as well as neurodegenerative disorders. The panel of caspase biosensors was utilized to investigate caspase cleavage specificity as well as caspase activation in mammalian cytosolic extracts and live mammalian cells. Perhaps more importantly, we discovered cross talk between members of the caspase family which perform different biological functions.Finally, we detail our progress towards mimicking a naturally occurring multicomponent complex formed during programmed cell death, known as the apoptosome which leads to the activation of caspases. We have successfully utilized principles of self assembly and multivalency to assemble multi component complexes which exhibit proteolytic activity similar to the natural apoptosome.

Eukaryotic protein kinases are one of the most important classes of human proteins, and a great deal of research has focused on the development of small molecule inhibitors as biological probes for the determination of their cellular function or as therapeutics for the treatment of disease, such as cancer. The need for new selective inhibitors and a better understanding of the selectivities of existing small molecules is readily apparent. Towards the goal of better understanding protein kinases and the molecules that inhibit them, I have developed a split-protein-based approach for the investigation of these kinase-small molecule interactions. Employing split-firefly luciferase as a reporter domain, we engineered a three-hybrid system capable of determining kinase inhibition through competitive interactions between an active site-directed ligand and a small molecule of interest. This method measures luciferase activity as a function of ligand binding, as opposed to the more traditional assays which quantify kinase activity directly, and alleviates the laborious process of protein purification. The model kinase PKA and the promiscuous ligand staurosporine were used in an initial test case to successfully validate the general design principles of our assay. The modular nature inherent to the assay's design enabled us to adapt it to roughly 300 additional protein kinases and two different ligands. We were able to establish a protocol for rapidly ascertaining the inhibition of a kinase by a library of 80 commercially available kinase inhibitors in a 96-well, high-throughput format. This protocol was then systematically applied to the AGC group of kinases to observe patterns of inhibition across similarly related kinases. We have further shown how these results might be correlated with the sequence identity between kinases to better anticipate inhibitor promiscuity. Finally, we were able to illustrate how a kinase-centric approach could be applied to correlate alterations to the kinase domain with changes in luminescence. This has use for the interrogation of different modes of inhibition as well as in identifying the specific determinants of inhibitor binding. In total, these efforts represent the optimization of a new, general platform for determining kinase inhibitor selectivity across the kinome, and it could potentially be applied universally to the interrogation of protein-ligand interactions.

The reversible phosphorylation of proteins plays a key role in nearly every aspect of cell life. This essential post-translational modification controls a myriad of cellular events from cell survival, differentiation, and migration to apoptosis. Two classes of enzymes, kinases and phosphatases, tightly control all phosphorylation events. Perturbation in the activity of any member of these classes of enzymes has been linked to numerous diseases including cancer, metabolic disorders, immune disorders and neurological disorders. Therefore, there is a great interest among the scientific community to develop methods to selectively modulate the activity of kinases and phosphatases not only for therapeutic purposes but also to understand the fundamental role of these enzymes in signaling events. The more than 500 kinases encoded in the human genome share a common catalytic fold and most inhibitors target the ATP binding site. Therefore selective targeting of a single kinase by an inhibitor at the highly conserved ATP binding site is one of the main concerns for designing probes or drugs. Our group has taken advantage of the potency and possible selectivity imparted by bivalent inhibitors and developed an in vitro selection approach to discover bivalent ligands. The strategy involves the use of an ATP-competitive small molecule warhead and a library of cyclic peptides displayed on phage that interact with the kinase of interest in a dynamic selection. The selection for a kinase binding peptide is carried out until consensus peptides are found and bivalent ligands are constructed by linking the selected cyclic peptide with the small molecule warhead through a synthetic linker. Using this approach a potent and selective bivalent inhibitor was found for PKA, a serine/threonine kinase. To interrogate the generality of this approach, a kinase closely related to PKA (PRKX) was used for which a very potent and selective bivalent ligand was found. The same selection strategy was further extended to the two kinases Lyn and Brk, which belong to the tyrosine kinase family. Though peptides were isolated that bound the desired kinase, potent bivalent inhibitors were not discovered. More generally, these experiments in sum are building a library of information regarding how to best design selections of potent and selective bivalent inhibitors. We further explored modulation of the activity of kinases and phosphatases by employing a ligand-gated split-protein approach. The small molecule gated spatial and temporal control of these enzymes should allow the study of signaling events in a controlled manner. The strategy employed consists in the identification of possible fragmentation sites within the catalytic domain of kinases and phosphatases by a sequence dissimilarity approach. Loop insertion mutants at the selected sites were tested for catalytic activity. Successful insertion mutants were further split into two catalytically inactive fragments, which were appended to two conditionally interacting protein domains. Upon addition of a small molecule, the two conditionally interacting domains reassemble the catalytic domain of the enzyme and restore catalytic activity. Using this approach we were able to modulate the activity of the tyrosine kinases Lyn, Fak and Src and the AGC kinase PKA. We also extended the approach to gate the activity of tyrosine phosphatases PTP1B, SHP1 and PTPH1. Finally, these ligand-gated split-kinases and phosphatases were validated in-cellulo. Thus, this work resulted in a new method for designing split-proteins and provided a palette of kinases and phosphatases that can be turned-on by small molecules. In total, these efforts describe two alternative routes that can be used to modulate phosphorylation events in a selective and controlled manner.

The specific interactions of macromolecules along with the activity of enzymes are central to all aspects of biology. It is well recognized that when the relative concentration or activity of macromolecules is perturbed, it can lead to human diseases. Thus, the development of simple methods for the detection of macromolecules and the activity of enzymes in complex environments is important for understanding biology. Moreover, the development of methods for measuring interactions allows for the testing of inhibitors that can be used as tools or drugs for improving human health. Towards this goal, a promising new method has been developed, which is the focus of this thesis, called split-protein reassembly or protein fragment complementation. In this method, a protein reporter, such as the green fluorescent protein or firefly luciferase, is dissected into two fragments, which are attached to designed adaptor proteins. The designed split-protein systems only produce a measurable signal, either fluorescence or luminescence, when a specific macromolecular interaction or activity is present. In this thesis, I have extended previous research on the direct detection of DNA using split-protein sensors utilizing a red fluorescent protein, dsRED from Discosoma that allows for multiplexed DNA detection. I have designed a new split-luciferase based sensor for detection of poly (ADP-ribose) or PAR, which plays a key role in the response to DNA damage and have applied it for monitoring the activity of poly (ADP-ribose) glycohydrolase that controls PAR levels in the cell. Furthermore, I have significantly expanded upon a three-hybrid split-luciferase system for identifying protein kinase inhibitors. I have designed and tested two orthogonal peptide based chemical inducers of dimerization based on BAD and p53mt conjugates. I have studied these chemically induced dimerization systems in detail in order to begin to provide a theoretical basis for the observed experimental results. Finally, in a less related area, I have developed methods for producing water soluble semiconductor nanoparticles called Quantum Dots (QDs), with potential application in biological imaging. I have developed methods for functionalizing the QDs with orthogonal peptides, which can be potentially used for the assembly of high affinity non-covalent QD targeted proteins.

The interactions between protein-protein, protein-nucleic acid, and protein-small molecules are central to biological processes and are key for the design of new therapeutics. Rapid and easy to implement methodologies are needed that enable the interrogation of these interactions in a complex cellular context. Towards this goal, I have utilized the concept of split-protein reassembly, also called protein complementation, for the creation of a variety of sensor architectures that enable the interrogation of protein-nucleic acid, protein-protein, and protein-small molecule interactions. Utilizing the enzymatic split-reporter β-lactamase and existing zinc finger design strategies we applied our recently developed technology termed SEquence-Enabled Reassembly (SEER) towards the creation of a sensor capable of the specific detection of the CryIA transgene. Additionally, the split β-lactamase reporter was utilized for the sitespecific determination of DNA methylation at cytosine residues that is involved in epigenetic regulation. This method, dubbed mCpG-SEER, enabled the direct detection of femtomole levels of dsDNA methylation in sequence specific manner. In a separate endeavor, we have developed and optimized the first cell-free split-reporter systems for GFP, split β-lactamase, and firefly luciferase for the successful dsDNA-dependent reassembly of the various reporters. Our cell free in vitro translation systems eliminates previous bottlenecks encountered in split-reporter technologies such as laborious transfection/cell culture or protein purification. Capitalizing on the ease of use and speed afforded by this new technology we describe the sensitive detection of protein-protein, protein-nucleic acid, and protein-small molecule interactions and inhibitors thereof. In a related area, we have applied this rapid cell-free split-firefly luciferase assay to the specific interrogation of a large class of helix-receptor protein-protein interactions. We have built a panel consisting of the clinically relevant Bcl-2 family of proteins, hDM2, hDM4, and p53 and interrogated the specificity of helix-receptor interactions as well as the specificity of peptide and small-molecule inhibitors of these interactions. Finally, we describe the further applications of our cell-free technology to the development of a large number of general split-firefly luciferase sensors for the detection of ssRNA sequences, the detection of native proteins, the evaluation of protease activity, and interrogation of enzyme-inhibitor interactions. The new methodologies provided in this study provides a general and enabling methodology for the rapid interrogation of a wide variety of biomolecular interactions and their antagonists without the limitations imposed by current in vitro and in vivo approaches.

Peptide fusion tags are fundamental for the identification, detection, and capture of proteins in biological assays. Commonly used peptide fusion tags rely on temporary non-covalent interactions for binding, which can put constraints on assay sensitivity. Here, peptide fusion tags were developed that could specifically interact with protein binding partners via spontaneous and irreversible isopeptide bond formation. To develop covalently interacting peptide-protein pairs, outer-membrane proteins from Gram-positive bacteria that form autocatalyzed intramolecular isopeptide bonds were dissected to generate a short peptide fragment and a protein binding partner. Initially, the major pilin subunit Spy0128 from Streptococcus pyogenes was split to develop the 16 residue isopeptag peptide and the 31 kDa pilin-C protein partner. The isopeptag:pilin-C pair were able to react via spontaneous isopeptide bond formation between an Asn residue in isopeptag and a Lys residue in pilin-C without the requirement for any accessory factors, and with a yield of 60% after a 72 hr reaction. Reconstitution between the isopeptag:pilin-C pair was robust and occurred under all biologically relevant conditions tested, and also in the complex environment of a bacterial cytosol and on the surface of mammalian cells. A similar approach was also used to dissect the small CnaB2 domain that is part of the large FbaB fibronectin-binding protein from S. pyogenes. This led to the development of a more efficient peptide-protein pair, which was rationally modified to generate the highly optimized SpyTag:SpyCatcher pair. SpyTag is a 13 amino acid peptide with a reactive Asp that forms a spontaneous intermolecular isopeptide bond with a Lys present in the 12 kDa SpyCatcher binding partner. In a reaction with SpyTag, over 40% of SpyCatcher was depleted after 1 min and SpyCatcher could no longer be detected after 2 hr. The SpyTag and SpyCatcher reaction did not require any accessory factors and proceeded efficiently at a range of biologically relevant temperatures, pH values, concentrations, buffer compositions, and in the presence of commonly used detergents. The SpyTag:SpyCatcher technology was also used for specific cell surface labelling on mammalian cell membranes. SpyTag and SpyCatcher are both composed of the regular 20 amino acids and can therefore be genetically encoded as fusion constructs for a variety of in vitro and in vivo applications. Potential applications of the SpyTag:SpyCatcher technology include specific cell surface labelling, the development of novel protein architectures, and the covalent and irreversible capture of target proteins in biological assays.

Peptide tags are ubiquitous in the life sciences, with roles including purification and selective labeling of proteins. Because peptide tags are small they have a limited surface area for binding and hence usually form low affinity protein interactions. These weak interactions limit the uses of peptide tags in cases that require resistance to forces generated with macromolecular architectures or protein motors. Hence a way to create a covalent interaction with a peptide tag would be useful. It was found possible to create a covalent bond-forming peptide tag using the spontaneous isopeptide chemistry of the CnaB2 domain from the Gram-positive bacterium Streptococcus pyogenes. In the CnaB2 domain a reactive Lysine forms an isopeptide bond with an Aspartic acid, catalyzed by a Glutamic acid, creating an internal covalent linkage. Subsequently it was shown that the CnaB2 domain could be split into two parts, a domain with the Lysine and Glutamic acid called SpyCatcher and a peptide with the Aspartic acid called SpyTag, such that the isopeptide covalent linkage can be formed when SpyCatcher/SpyTag are mixed together. SpyCatcher/SpyTag was applied in this thesis and showed functionality in a wide array of scenarios. SpyCatcher/SpyTag covalently linked within the cytosol of E. coli, on surface membrane proteins of HeLa cells, and regardless of whether SpyTag was located on the N- or C-terminus or an internal site. Crystal structures of SpyCatcher/SpyTag were then obtained and it was found possible to shrink the SpyCatcher by 32 residues to a core domain of 83 residues. To create an even smaller covalent linkage system, SpyCatcher was split further to generate a protein (SpyLigase) ligating two peptide tags. The β-sheet with the reactive Lysine was removed from SpyCatcher and called KTag. SpyLigase could covalently link SpyTag and KTag. SpyLigase-induced ligation was independent of the location of SpyTag/KTag on the target proteins and was applied to create affibody polymers, which were shown to improve magnetic isolation of cells with low tumor antigen expression. Through this work protein-protein covalent linkage systems were refined and generated that have future applications for the creation of unique macromolecular structures, cellular labeling, and protein cyclization.