Thesis advisor: Evan R. Kantrowitz / The regulatory mechanism of allostery is exhibited by certain proteins such as Escherichia coli aspartate transcarbamoylase (ATCase), and is defined as the change in shape and activity (of enzymes) resulting from the binding of particular molecules at locations distant from the active site. This particular enzyme and the property of allostery in general have been investigated for several decades, yet the molecular mechanisms underlying allosteric regulation remain unclear. Therefore in this thesis we have attempted via several biophysical methods, along with the tools of molecular biology and biochemistry, to correlate the changes in allosteric structure with presence of the allosteric effectors and enzymatic activity. We created a double mutant version of ATCase, in which the only native cysteine residue in the catalytic chain was mutated to alanine and another alanine on a loop was mutated to cysteine, in order to lock the enzyme into the R allosteric state by disulfide bonds. This disulfide locked R state exhibited no regulation by the allosteric effectors ATP and CTP and lost all cooperativity for aspartate, and then regained those regulatory properties after the disulfide links were severed by addition of a reducing agent. This double mutant was then chemically modified by covalent attachment of a fluorescent probe. The T and R allosteric states of this fluorophore-labeled enzyme had dramatically different fluorescence emission spectra, providing a highly sensitive tool for testing the effects of the allosteric effectors on the allosteric state. The changes in the fluorescence spectra, and hence quaternary structure, matched the changes in activity after addition of ATP or CTP. This fluorophore labeled enzyme was also encapsulated within a solgel, changing the time scale of the allosteric transition from milliseconds to several hours. The fluorophore labels allowed monitoring the allosteric state within the sol-gel, and the physically trapped T and R states both showed no regulation by the allosteric effectors ATP and CTP, and no cooperativity for aspartate. The trapped T state had low-affinity for aspartate and low activity, and the trapped R state had high-affinity for aspartate and high activity. Timeresolved small-angle x-ray scattering (TR-SAXS) was used to determine the kinetics of the allosteric transition, and to monitor the structure of the enzyme in real time after the addition of substrates and allosteric effectors. These TR-SAXS studies demonstrated a correlation between the presence of the allosteric effectors, the quaternary allosteric state, and activity, suggesting like the previous studies in this thesis that the behavior of ATCase is well explained by the twostate model. However, the effector ATP appeared to destabilize the T state and CTP to destabilize the R state, suggesting a different allosteric molecular mechanism than that of the two-state model. This thesis demonstrates the validity of many of the concepts of the two-state model, while suggesting minor modifications to that elegantly simple model in order to conform with the complex structure and function of ATCase. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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The homologous p10 fusion-associated small transmembrane (FAST) proteins of the fusogenic avian (ARV) and Nelson Bay (NBV) reoviruses are the smallest known proteins capable of mediating syncytiogenesis. Their extremely small size precludes them from following the paradigmatic membrane fusion pathway proposed for enveloped viral fusion proteins. I exploited the sequence conservation/divergence and differential syncytiogenic rates between ARV and NBV to define functional motifs in the p10 ectodomains. Using chimeric p10 constructs, I determined the 40-residue ectodomain (sizes refer to ARV) comprises two distinct functional motifs essential for syncytiogenesis. Cellular syncytiogenic and surface biotinylation assays identified an indivisible, 25- residue, N-terminal ectodomain motif required for cystine loop fusion peptide formation. I further determined the roles of this cystine loop in promoting lipid binding and cholesterol-dependent lipid destabilization. Immunofluorescence staining, FRET analysis and cholesterol depletion/repletion studies identified a second motif comprising the 13 membrane-proximal ectodomain residues (MPER). This motif governs the reversible, cholesterol-dependent assembly of p10 multimers in the plasma membrane. I demonstrate that ARV and NBV homomultimers segregate to separate foci in the plasma membrane, and the four juxtamembrane residues present in the multimerization motif dictate species- specific homomultimerization. I also discovered the novel codependency of p10 multimerization and cholesterol-dependent microdomain localization. The majority of enveloped virus membrane fusion proteins function as stable multimers, which nonetheless must undergo dramatic, irreversible, tertiary structure rearrangements to mediate membrane fusion. Cholesterol-rich membrane microdomains have also been implicated in the function of several enveloped virus fusion proteins, and a limited number of studies have investigated the role of cholesterol in multimerization. My results reveal cholesterol-dependent p10 homomultimerization is an essential aspect of p10- mediated syncytium formation, and I identify the motifs responsible for this process. The reversible nature of p10 cholesterol-dependent multimerization at the plasma membrane is in line with several other studies suggesting that the dynamic clustering and dispersion of cholesterol microdomains, as well as protein transitioning from multimeric to monomeric intermediates, are essential phenomena of protein mediated membrane fusion.

Kinases are involved in a multitude of signaling pathways, such as cellular growth, proliferation, and apoptosis, and have been discovered to be important in numerous diseases including cancer, Alzheimer's disease, cardiovascular health, rheumatoid arthritis, and fibrosis. Due to the involvement in a wide variety of disease types, kinases have been studied for exploitation and use as targets for therapeutics. There are many limitations with developing kinase target therapeutics due to the high similarity of kinase active site composition, making the utilization of new techniques to determine kinase exploitability for therapeutic design with high specificity essential for the advancement of novel drug strategies. In silico approaches have become increasingly prevalent for providing useful insight into protein structure-function relationships, offering new information to researchers about drug discovery strategies. This work utilizes streamlined computational techniques on an atomistic level to aid in the identification of orthologue and isoform exploitability, identifying new features to be utilized for future inhibitor design. By exploring two separate kinases and kinase targeting domains, we found that orthologues and isoforms contain distinct features, likely responsible for their biological roles, which can be utilized and exploited for selective drug development. In this work, we identified new exploitable features between kinase orthologues for treatment in Human African Trypanosomiasis and structural morphology differences between two kinase isoforms that can potentially be exploited for cancer therapeutic design. / Master of Science in Life Sciences / Numerous diseases such as cancer, Alzheimer's disease, cardiovascular disease, rheumatoid arthritis, and fibrosis have been attributed to different cell growth and survival pathways. Many of these pathways are controlled by a class of enzymes called kinases. Kinases are involved in almost every metabolic pathway in human cells and can act as molecular switches to turn on and off disease progression. Due to the involvement of these kinases' in a wide variety of disease types, kinases have been continually studied for the development of new drugs. Developing effective drugs for kinases requires an extensive understanding of the structural characteristics due to the high structural similarity across all kinases. In silico, or computational, techniques are useful strategies for drug development practices, offering new information into protein structure-function relationships, which in turn can be utilized in drug discovery advancements. Utilizing computational methods to explore structural features can help identify specific protein structural features, thus providing new strategies for protein specific inhibitor design. In this work, we identified new exploitable features between kinase orthologues for treatment in Human African Trypanosomiasis and structural morphology differences between two kinase isoforms that can potentially be exploited for cancer therapeutic design.

Pentameric ligand-gated ion channels (pLGICs) are membrane-embedded receptors found extensively in pre- and post-synaptic membranes throughout the nervous system where they play an important role in neurotransmission. The function of the prototypic pLGIC, the nicotinic acetylcholine receptor (nAChR) is highly sensitive to changes in its lipid environment, while other pLGICs display varying lipid sensitivities. This thesis presents a multidisciplinary investigation into the features of the transmembrane domain (TMD) that determine the unique functional and physical traits of different pLGICs. Using two prokaryotic homologues of the nAChR, ELIC and GLIC, as models, I focus on the outermost, lipid-exposed α-helix, M4, which, despite being distant from the primary allosteric pathway coupling agonist binding to channel gating, exercises significant control over channel function. Here, I present evidence that M4 acts as a lipid sensor, detecting changes in the surrounding lipids and transmitting these changes to the channel pore via contacts with the adjacent TMD α-helices, M1 and M3, and/or with structures in the extracellular domain. Using ELIC and GLIC chimeras, I first show that the TMD is the main driver of pLGIC thermal stability. I then demonstrate that the M4 α-helices in each channel play different roles in channel maturation and function, which suggests a divergent evolutionary path. Following this, I show that the M4 C-terminus is essential to both maturation and function in GLIC, while in ELIC its role is less defined, again showcasing possible evolutionary differences. Building on these findings, I examined the role of aromatic residues at the M4 – M1/M3 interface, and found that they predictably determine the interactions between M4 and M1/M3. Notably, the addition of aromatic residues to enhance M4-M1/M3 interactions in ELIC promotes channel function, while the elimination of aromatic residues at the M4-M1/M3 interface in GLIC is detrimental to channel function. Furthermore, I show that these same aromatics alter the strength of pLGIC lipid sensing and the sensitivity to certain disease-causing mutations, both indicating that aromatic residues are key players in channel function, stability and modulation. Finally, I and my collaborators identified and characterized a novel desensitization-linked lipid binding site in ELIC. Extensive mutagenesis studies coupled with biophysical measurements allowed us to develop a model describing how lipid binding influences the rates of ELIC desensitization to shape the agonist-induced response.