ItemAntifreeze Protein EngineeringStevens, Corey; Biochemistry; Davies, PeterThis thesis describes: 1) the use of protein engineering to increase ice-binding protein (IBP) activity and thermal stability, and 2) the binding interaction and microcolony formation between an Antarctic bacterium and diatom. IBPs, including the antifreeze proteins (AFPs) that prevent the freezing of organisms, are found in nearly all biological kingdoms. IBPs have potential applications in a variety of domains including the food industry, cryo-medicine, and biotechnology. Despite the variety of IBPs, most are difficult to produce in amounts needed for industrial applications. Consequently, there is a need to find or engineer IBPs with enhanced activity and stability. Previously, AFP activity was increased by fusing an AFP to another protein, or by increasing the size of the IBP’s ice-binding face. Here, I used a highly-branched polymer, known as a dendrimer, to fuse a range (6 to 12) of moderately-active type III AFPs from Macrozoarces americanus together. These AFP multimers had improved antifreeze and ice-recrystallization inhibition activity. Unexpectedly, AFPs multimers had enhanced recovery from heat treatment. I also achieved enhanced thermal stability in type III AFP through an alternative strategy. Using split-intein mediated end-terminal ligation, I fused the N- and C- termini of the type III AFP together. Peptide backbone circularization had no effect on antifreeze activity but significantly increased thermal stability compared to the non-cyclized form. The IBP found on the cell surface of a Gram-negative Antarctic bacterium, Marinomonas primoryensis, is one region of an exceptionally large multi-domain 1.5 MDa protein, MpIBP. Using temperature-controlled microfluidics, I have shown that M. primoryensis forms bacterial clusters on ice. Binding is aided by the motility of the bacterium and is dependent on the functionality of its ice-binding domain. The strictly aerobic M. primoryensis is drawn and binds to, the Antarctic diatom Chaetoceros neogracile to form mixed cell clusters and adheres them to ice. We hypothesize that the ice-binding function of MpIBP keeps its host immediately under the surface ice in the phototrophic zone of the water column where oxygen and carbon compounds are more abundant. By recruiting diatoms to the ice, M. primoryensis helps these photosynthesizers form a symbiotic community where light is most abundant. ItemStructural and Functional Characterization of the Human Tribbles Homologue 2 Pseudokinase(2016-10-27) Hicks, Gregory; Biochemistry; Jia, ZongchaoThe Tribbles Homologues are a family of three eukaryotic pseudokinases (Trb1, Trb2, Trb3) that act as allosteric inhibitors and regulatory scaffold sites in pathways governing adipogenesis, cell proliferation and insulin signaling. The Tribbles Homologues have the same overall tertiary structure of the eukaryotic protein kinase domain, but lack multiple residues necessary to catalysis in the nucleotide-binding P-loop and the Mg2+-coordinating DFG motif. Trb1 has been shown conclusively to be incapable of binding ATP, whereas a recent study presents evidence that Trb2 autophosphorylates independently of Mg2+ in vitro. This finding is surprising given the high degree of sequence similarity between the two proteins (71%), and suggests unique nucleotide binding and phosphotransfer mechanisms. The goal of this project was to investigate whether Trb2 possesses kinase activity or not and determine its structural basis. A method for the high-yield recombinant expression and purification of stable Trb2 was developed. Trb2 nucleotide binding and autophosphorylation could not be detected across multiple experimental approaches, including thermal shift assays, MANT-ATP fluorescence, radiolabeled phosphate incorporation, and nonspecific ATPase activity assays. Further characterization also revealed that Trb2 forms homomultimers with possible functional consequences, and extensive crystallization screening has yielded multiple promising conditions that could produce diffraction-quality crystals with further optimization. This project explores the difficulties in functionally characterizing putatively active pseudokinases, and proposes a structural basis for the conserved pseudokinase features of the Tribbles homologues. ItemInvestigating the Role of PDE4D1/2 in Vascular Smooth Muscle Cell Desensitization(2016-10-14) Butler, Nathalie; Biochemistry; Maurice, Donald H.Vascular smooth muscle cell (VSMC) behaviour and phenotypic modulation is critical to vessel repair following damage, and the progression of various cardiovascular diseases. The second messenger cyclic adenosine monophosphosphate (cAMP) plays a key role in VSMC function under the synthetic/activated phenotype, which is typically associated with unhealthy cell behaviour. Consequently, cAMP signaling is often targeted in attempts to impact several pathological diseases, including atherosclerosis, restenosis, and pulmonary arterial hypertension (PAH). The cyclic nucleotide phosphodiesterases (PDEs) catalyze hydrolysis of cAMP to an inactive form, and therefore directly regulate cAMP signaling. The PDE4D family dominates in synthetic VSMCs, and there is considerable interest in determining how distinct PDE4D isoforms affect cell function. Specifically, we are interested in the potential link between short isoforms of PDE4D and VSMC desensitization to pharmacological agents that impact cardiovascular disease via cAMP signaling. This study extends on previous work that assessed the expression of PDE4D splice variants in rat aortic VSMCs following prolonged challenge with cAMP-elevating agents. It was determined that PDE4D1 and PDE4D2 were uniquely expressed in synthetic VSMCs incubated with these agents, and that this upregulation impacted PDE activity and cAMP accumulation in these cells. Here, we report that PDE4D1 and PDE4D2 are markedly upregulated in synthetic human aortic smooth muscle cells (HASMCs) following prolonged challenge with cAMP-elevating agents. Using a combination of RNAi-based and pharmacological approaches, we establish that this upregulation is reflected in levels of cAMP PDE activity, and restricted to the cytosolic sub-cellular compartment. Our results suggest a role for localized PDE4D1 and PDE4D2 activity in regulating cAMP-mediated desensitization in HASMCs, and highlight their therapeutic potential in treating various cardiovascular diseases. ItemDiscovery and characterization of an antifreeze protein from Lake Ontario midges(2016-05-02) Basu, Koli; Biochemistry; Davies, Peter L.This thesis describes the discovery and characterization of the first antifreeze protein (AFP) isolated from a fly. The starting point of my research was the observation that when midges emerge from Lake Ontario as adults in early spring they have low levels of antifreeze activity. Here I have isolated, characterized, and modeled the structure of their main AFP. This 79-residue mature midge AFP has a novel sequence of ten-residue tandem repeats, xxCxGxYCxG, which I modeled as is a left-handed disulfide-braced solenoid, with each 10-residue repeat corresponding to one coil of the helix. The fold is similar to a beta-helix, but secondary structure and circular dichroism analyses indicate that this solenoid is too tightly coiled to have beta-structure. The model shows an outward pointing seven-residue stacked Tyr-ladder, which has been confirmed by mutagenesis to serve as its ice-binding site. This is the first example of tyrosines used for ice binding. I have determined that the midge AFP activity is intermediate to the moderately active and hyperactive AFPs typically found in fish and overwintering insects, respectively. The proposed explanation for intermediate activity is that the midge AFP binds to a pyramidal surface midway between the basal and prism planes. The modest sub-zero temperatures that the adult flies encounter has likely driven the evolution of this intermediate activity AFP. I predict that other organisms facing freezing threats of a similar magnitude will also produce AFPs with intermediate activity. I have also contributed to the literature on experimental methods used to assess ice-binding properties of AFPs by publishing a step-by-step protocol for the fluorescence-based ice-plane affinity assay with an instructional video. This technique can be used, as it was here, to determine which planes of ice are bound by fluorescently-labelled AFPs. A second chapter on techniques describes the ab initio and homology-based techniques our lab has used to reliably predict novel ice-binding protein folds. ItemStructure-Guided Studies of Bacterial Competition Mechanisms(2015-12-16) Podzelinska, Kateryna; Biochemistry; Jia, ZongchaoMicroorganisms have evolved a stunning array of strategies for nutrient competition, ranging from concerted effort of antibiotic release to kill off competing species, to evolving complex enzymatic pathways that are capable of scavenging nutrients from sources not utilizable by other organisms. The carbon-phosphorous (C-P) lyase pathway is a survival mechanism that is activated during phosphate limitation in certain species of bacteria and enables cleavage of the extremely stable C-P bond in order to obtain phosphorous from organophosphonates. The structure and biochemical characterization of PhnP, a critical accessory protein from C-P lyase pathway of E. coli is presented in this thesis. The structure of PhnP revealed a conserved metal-dependent hydrolase active site with two Mn2+ ions, and another unique mononuclear Zn2+ site that appears to have a structural role. A non-physiological ligand that fortuitously co-crystallized with the enzyme provided insights into the catalytic features of the active site. We were able to demonstrate hydrolytic activity towards a number of phosphodiesterase substrates, and propose a plausible physiological role for PhnP. These results contribute to deciphering the mechanism of phosphonate utilization, which would allow design of bioremediation programs to remove toxic phsophonates from the environment. Antibiotic production is another mechanism for resource competition. Structural characterization of CmlS, a halogenase from the chloramphenicol biosynthesis pathway of S. venezuelae is presented. The crystal structure revealed a novel covalent modification of its FAD cofactor, which was confirmed through ESI-MS and chemical denaturation studies. The unique C-terminal domain, active site architecture, and the position of the C-terminus suggest that halogenation mechanism of CmlS may differ from the currently proposed mechanism for structurally related halogenases. This work provides early steps towards understanding mechanisms of enzymatic halogenations, which is of great scientific, as well as pharmaceutical interest.