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Two main Arabidopsis thimet oligopeptidases (AtTOP) involved in stress responses are: (1) thimet metalloendopeptidase 1 (TOP1), found in the mitochondria and chloroplasts, annotated as At5g65620, and (2) thimet metalloendopeptidase 2 (TOP2), found in the cytosol annotated as At5g10540. Both AtTOP1 and AtTOP2 are located on chromosome 5 and share high homology. AtTOP1 and AtTOP2 are zincin-like metalloendopeptidases with the characteristic HEXXH active motif of the M3 clan. Their peptidase activity is related to the oxidative stress triggered by plant immune responses. AtTOPs are involved in plant immune responses through a mechanism regulated by Salicylic Acid (SA); both AtTOP1 and AtTOP2 bind plant SA, which inhibits their peptidase activities. However, we engineered a series of mutations to identify which cysteines are responsible for TOPs dimerization and other oxidative, structureunction related events. Each of the cysteine in TOPs (i.e., six cysteines in TOP1, and four cysteines in TOP2) were independently mutated to alanine, as a single mutant. The dynamics of the oxidative dimerization processes were measured using gel filtration and native gel methods to quantify the dimerization process of both native and mutant TOPs under variable redox potentials ex vivo and in vitro at various GSH/GSSG and DTTox/DTTred ratios, with the underlying hypothesis that the TOP dimerization and enzymatic activities are regulated by changes in the disulfide bond formation that is linked to cellular redox environments. Overall our results indicate that TOP1 is sensitive to changes in the redox environment, while TOP2 is not. The monomer/dimer ratio of TOP1 in solution is higher under highly reducing conditions compared to mildly and highly oxidative environments. Two TOP1 cysteines control the formation of dimers, one located in its N-terminal signal peptide (C52) and the other located in the peptidase domain (C611). These findings bring a mechanistic understanding of TOP1 and TOP2 functions in the plant immune response.

Thesis advisor: Eranthie Weerapana / This thesis includes two projects: “Bacteria-selective borono-peptides” and “A split ligand for lanthanide binding: facile evaluation of dimerizing proteins”. In both projects, de novo designed molecules were synthesized, optimized and incorporated into peptides. These synthetic molecular tools allow selective targeting of bacterial cell membranes and analyzing the dynamic associations of membrane-embedded proteins. 1. Bacteria-selective borono-peptides As the antibiotic resistance continues to grow, bacterial infection becomes one of the major threats to global public health. Currently, almost all the bacteria targeting strategies employ non-covalent driving forces, including charge-charge interactions, hydrophobic interactions and the formation of hydrogen bonds, to achieve bacterial selectivity. Towards novel bacteria targeting molecules, we have recruited reversible covalent chemistry in the development of bacteria-selective peptides. Targeting the diol-rich environment of a bacterial surface, we have designed and synthesized several unnatural amino acids that contain boronic acid moieties. Taking advantage of the boronic acid-diol reaction and multivalency effect, our borono-peptides are found to selectively recognize bacteria over mammalian cells. The sensitivity of the binding event to carbohydrate competitors gives a safe and facile approach to regulate molecular association with bacterial cells. This design may find applications in the fields of bacterial detection, imaging and antimicrobial drug delivery. 2. A split ligand for lanthanide binding: facile evaluation of dimerizing proteins Protein dimerization is a ubiquitous phenomenon in biology and plays a critical role in transcription regulations and various signaling processes. Methods that allow facile detection and quantification of protein dimers are highly desirable for evaluating protein dimerization in physiology and disease. Meanwhile, luminescence of lanthanides is attractive for biological applications due to its long lifetime and sharp emission profiles. We have developed a split lanthanide binding ligand that allows facile evaluation of dimerizing proteins. The fast lanthanide–ligand (dis)association allows us to monitor the dynamic behavior of dimerizing proteins. We have demonstrated the successful application of our assay on both soluble and transmembrane proteins in complex biological milieu. The split lanthanide ligand is cysteine reactive, and therefore should be readily applicable to a variety of proteins of interest. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Group II introns are highly structured RNAs that carry out self-splicing reactions. The multiple turnover version of one of these introns, termed the D135 ribozyme, is derived from the mitochondrial aI5γ intron of Saccharomyces cerevisiae and is widely studied as a model RNA for group II intron folding. An important current goal is to probe global changes during its folding with or without DEAD-box chaperone proteins. My initial experiments to study global compaction using small angle X-ray scattering (SAXS) of D135 reveal rapid initial compaction. Unexpectedly, slower increases in Rg value and forward scattering were observed and shown to result from dimerization of the ribozyme. Dimerization was also observed with native electrophoretic mobility shift assays. Here, I have characterized the dimerization process at various conditions. Dimerization requires Mg2+, with similar concentration dependence as tertiary folding, and the dimer is efficiently disrupted by the ATP-dependent activity of DEAD-box proteins. Dimerization does not affect ribozyme catalysis, as both the monomer and the dimer are shown to be fully active. Further experiments showed that dimerization results from duplex formation by an artificial 3’ tail that has extensive self-complementarity, as the deletion of this tail ablates dimerization. Constructs lacking this artificial 3’ tail are likely to simplify further study of the folding process of this ribozyme. / text

Chemical inducers of dimerization (CID) represent an important tool that has been implemented in numerous biological applications namely protein functions, protein stability, signal transduction, gene transcription, etc. Most generally CIDs are defined as bivalent molecules capable of inducing proximity between two targeted proteins. This proximity can in turn promote or disfavor a certain biological activity. Cell permeable small molecules in particular represent a very effective method to induce precise temporal and spatial control over a specific biological target. Our lab has devoted much effort in studying and elucidating the activity and functions of protein kinases, which represent a very attractive therapeutic target for the treatment of cancer and many other disorders. Towards this goal we have developed a general CID enabled three-hybrid split-luciferase methodology for the investigation of kinase-inhibitor interactions in vitro. We demonstrate that by modulating the kinase-ligand affinity of the CID we are able to successfully profile many structurally non-related protein kinases. We also investigate the use of weaker affinity kinase ligands to allow competitive displacement of CID by the selected inhibitor. In addition we report the design, synthesis and applications of novel CID's for the profiling of kinase inhibitors in mammalian cells and we demonstrate the feasibility of the assay to be used as a new platform for the discovery of cell permeable kinase inhibitors. Finally, we report a new ligand-gated split-kinase that can be selectively activated by photocleavable inducers of dimerization. We further prove how the activity of split-proteins can be deactivated with temporal control with use of non DNA damaging UV radiation.

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Thesis advisor: Jianmin Gao / The oligomerization of membrane proteins has been shown to play a critical role in a myriad of cellular processes, some of which include signal propagation, cell-to-cell communication, and a cell's ability to interact with its surroundings. Diseases that are associated with disruption of protein-protein interactions in the membrane include cystic fibrosis, certain cancers, and bone growth disorders. Although significant progress has been made in our mechanistic understanding of protein-protein interactions in membranes, it remains difficult to predict the oligomerization state of transmembrane domains and explain the physiological consequences of a point mutation within a membrane embedded protein. The development of novel classes of chemical tools will allow us to better understand the energetics of transmembrane domain association at the molecular level. Herein, we demonstrate that fluorinated aromatic amino acids offer intriguing potential as chemical mediators of transmembrane protein association. We have systematically examined the effects of fluorination on the physical properties of aromatic systems in the context of a soluble protein model system. Our results illustrate the ability of fluorinated aromatic amino acids to simultaneously stabilize protein structure and facilitate highly specific protein self-assembly. An improved understanding of the fundamental energetics of aromatic interactions should allow for their more efficient incorporation into designed inhibitors of transmembrane protein association. In addition to chemical tools, the development of simple methods for directly monitoring transmembrane domain association in vitro and in vivo is necessary to advance our understanding of these interactions. Towards this goal, we have established FlAsH-tetracysteine display as an effective approach to quantifying the association propensities of transmembrane α-helices (TMHs) in vitro. Our assay is compatible with two of the most commonly utilized model membrane systems, detergent micelles and vesicles. The high spatial resolution of FlAsH binding (˂ 10 Å) allows for the differentiation of parallel and antiparallel oligomerization events. Importantly, preliminary studies suggest the assay's ability to detect inhibition from exogenous TMHs. Encouraged by our understanding of aromatic interactions and the success of our assay, we are beginning to incorporate fluorinated aromatics in the model TMHs and monitoring their ability to associate. The ultimate goal is to modulate the association of endogenous TMHs such as ErbB2. Research in this direction is ongoing. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Nickel insertion into the [NiFe]-hydrogenase requires the accessory protein HypB, which is a GTPase. The GTPase domain of Escherichia coli (E. coli) HypB undergoes dimerization in the presence of GTP. To determine the role of HypB dimerization in hydrogenase biosynthesis, a double mutation L242A/L246A was introduced into full-length E. coli HypB, and the protein was expressed and characterized both in vitro and in vivo. Gel filtration experiments demonstrated that L242A/L246A HypB was monomeric as expected. The inability of L242A/L246A HypB to dimerize does not abolish its GTPase activity and the monomeric L242A/L246A HypB has a similar Ni(II)-binding behavior as that of wild type HypB. Upon the expression of L242A/L246A HypB in vivo the hydrogenase activity is approximately half of the activity of the wild-type control. These experimental results suggest that dimerization of HypB does have a, but not critical, role in hydrogenase biosynthesis.