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Soluble enzymes have the unique ability to assist the conversion of complex substrates in fewer steps compared to conventional chemical synthesis; however, when immobilized to a surface, the insoluble enzyme's activity and stability can suffer in its new environment. To improve upon future immobilized enzyme efficacy, a fundamental understanding of the interactions across the enzyme, linker, and surface during covalent, site-specific immobilization that can be detrimental to its activity is required. More specifically, using a well-characterized and model enzyme, T4 lysozyme (T4L), 28 different cysteine attachment points' effects on site-specifically immobilized activity are examined. Different combinations of heterobifunctional amine-to-sulfhydryl crosslinkers and hydrophobic and hydrophilic aminated polymer surfaces are used to immobilize T4L site-specifically via amine-to-sulfhydryl conjugation. The cysteine mutations availability and accessibility result in heuristics that minimize nonspecific adsorption while maximizing site-specific conjugation. Based on the findings, an improved method for site-specifically immobilizing biotinylated T4L on streptavidin-coated polymer beads is developed. Innate structural characteristics such as cysteine accessibility, amino acid consensus, and enzyme surface concentration enhance immobilized enzyme’s specific catalytic efficiency regardless of the attachment site and orientation.

Chapter 1. In the Tan laboratory, we developed synthetic methods to control reaction selectivity (regio-, stereo-, and site-selectivity) using scaffolding catalysis. Our strategy utilizes directing groups that induce intramolecularity through the formation of a labile covalent bond between the substrate and a binding site in a catalytic system. In the first part, we described site-selective functionalization of various carbohydrates and complex polyhydroxylated molecules which contain cis-1,2-diol motif using a chiral organic scaffold. In the second part, meta-selective C–H functionalization of arenes was demonstrated. High meta-selectivity was achieved by the use of a nitrile-based silyl tether which is cleavable and recyclable. Chapter 2. In the Liu laboratory, we focuses on studies of boron-nitrogen containing heterocycles. In this chapter, synthesis of 1,2-azaborines and their binding study with T4 lysozyme mutants were described. Specifically, we directly compared binding of NH-containing 1,2-azaborines and their carbonaceous analogs to probe hydrogen bonding interaction between the NH group of azaborine and a carbonyl oxygen of protein residue. Structural and thermodynamic analysis provided us the first evidence of H-bonding of azaborines with a biological macromolecule. Chapter 3. Described are the synthesis of regioisomers of ethyl-substituted 1,2-azaborines and their binding thermodynamics to T4 lysozyme mutants. To access the azaborine ligands used in the binding study, we developed synthetic methods for regioselective functionalization of six positions of 1,2-azaborines. Isothermal titration calorimetry experiments showed differences in binding free energy for regioisomers to the L99A T4 lysozyme. This result could originate from electronic differences of the isosteric ligands inducing dipole-dipole interaction between ligand and surrounding protein residues or it may be from local dipolar interactions. Chapter 4. A general method for late-stage N-functionalization of 1,2-azaborines is described to afford libraries of BN-containing complex molecules. The chemical transformations include electrophilic substitution reactions, N–C(sp2) bond forming reactions under Buchwald-Hartwig amination conditions, and N–C(sp) bond forming reactions using copper-catalyzed N-alkynylation. As applications in materials science and medicinal chemistry, synthesis of the first parental BN isostere of trans-stilbene and lisdexamfetamine derivative is described utilizing the methodology developed in this work.

Described in this thesis is the use of controlled radical polymerization techniques to synthesize tetrazine end-functionalized telechelic polymers, which were employed to generate T4 Lysozyme homodimers. A mutant of T4 Lysozyme (V131C), containing a single surface-exposed cysteine residue, was modified via Michael addition with a protein-reactive trans-cyclooctene. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to obtain telechelic poly(N-isopropylacrylamide) (pNIPAAm) polymers with Mn (1H-NMR) of 2.0 kDa and molecular weight dispersity, Đ, (GPC) of 1.05. pNIPAAm was modified at both ends with (4-(6- methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanol. As a control, a 2.0 kDa bis-carboxylic acid poly(ethylene glycol) was modified at both ends with (4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl)methanol and a 2.0 kDa bis-maleimide pNIPAAm was also synthesized. Tetrazine- functionalized polymers were then ligated to a trans-cyclooctene modified Lysozyme (T4L-TCO). Ligation of T4L-TCO to bis-tetrazine pNIPAAm resulted in 27.27% yield of the homodimeric conjugate, whereas bis-tetrazine PEG resulted in 16.75% and bis-maleimide pNIPAAm resulted in

Proteins in solution exhibit structural fluctuations on a wide range of characteristic time scales, from fast backbone motions in the picosecond to nanosecond range to slow conformational exchange in the microsecond to millisecond time domain. These motions can play important roles in function, thus elucidation of molecular mechanisms underlying function requires experimental techniques capable of measuring motions on these time scales. Site directed spin labeling and EPR spectroscopy (SDSL-EPR) has been established as an important tool in protein science for studying structure, identifying dynamically disordered sequences, and monitoring conformational switching triggered by chemical or physical signals in soluble and membrane-bound proteins. However, it remains to be investigated whether fast backbone motions contribute to the spectra of the spin label side chain R1 in well-ordered sequences of regular secondary structure, and how slow conformational exchange between substates can be detected at equilibrium; these are goals of this dissertation. The results from this work support a model wherein variations in the EPR spectral lineshape of R1-labeled proteins measures the amplitude of fast but constrained backbone fluctuations on the nanosecond time scale, and show that these motions are correlated with the local packing density. In addition, it is shown that osmolyte perturbation SDSL (OP-SDSL) can be used to identify conformational equilibria between substates that have characteristic lifetimes > 100 ns. The newly-developed OP-SDSL strategy was employed along with continuous wave (CW) lineshape analysis to map molecular flexibility of sperm whale myoglobin in folded and partially folded states and of core-repacking mutants of T4 lysozyme. For myoglobin, the results reproduce and extend earlier NMR studies, thus validating the OP-SDSL method and highlighting advantages of the inherent EPR time scale. For T4 lysozyme, it is found that core repacking mutations that generate internal cavities can give rise to new conformational substates in solution, despite the fact that earlier studies show that the corresponding crystal structures of WT and mutants were essentially identical. The results also show that ligand binding to some of the engineered cavities dramatically shifts the populations of substates towards the native-like state. Collectively, the results of this research advance the SDSL-EPR methodology for facile mapping of protein flexibility over the important picosecond to millisecond time domain and demonstrate the utility of the technology for exploring the molecular basis of protein function.

Lettuce crop is affected by bacterial diseases (both in the field and in storage) with severe, consequences which can be mitigated through improving disease resistance in lettuce. The numerous constraints associated with sexual crosses in lettuce render novel gene transfer via genetic manipulation a suitable method for lettuce improvement. This study was undertaken to transfer three chimaeric genes encoding antibacterial peptides into lettuce and to ascertain the effectiveness of these peptides in enhancing bacterial disease resistance. Genetic manipulation of plants requires a good regeneration ability of genotypes in tissue culture, to ensure recovery of complete transformed plants. Twenty-two lettuce genotypes belonging to several morphological groups were thus screened for their regeneration response to defined tissue culture conditions and selection of amenable genotypes for genetic manipulation. Genotypic variation was observed in callus, shoot and root production in vitro; two lettuce genotypes Bambino (a crisphead) and Cobham Green (a butterhead) with good tissue culture response were subsequently chosen for Agrobacterium-mediated transformation. Two binary vectors carrying chimaeric genes (with T4 lysozyme, magainin IT coding sequences) were obtained from Crop & Food Research, Lincoln; these vectors had pBINPLUS (a recently constructed binary plasmid) as the backbone. A third binary vector, similar in structure to the two others, with a chimaeric gene encoding Shiva-1 (a cecropin B analogue) was successfully constructed. The three vectors, pBINPLYS, pBINMGN and pBINPLUSH (encoding T4 lysozyme, magainin IT and Shiva-1 respectively) in Agrobacterium strain AGLI were used for the transformation of Bambino and Cobham Green. Two transformation treatments: a delay of 5 days (after co cultivation) before explants were transferred to kanamycin selection medium and an immediate transfer of explants to selection medium were studied for their effect on transformation frequency. Successful regenerated transformed plants of Cobham Green were obtained in this study; however, attempts to transform Bambino proved futile and no regenerated transformed shoots of Bambino were obtained. The specific reasons for the failure to transform Bambino are unknown but crisphead lettuce genotypes are often recorded as being recalcitrant to Agrobacterium-mediated transformation. The delayed selection treatment gave higher transformation frequencies in Cobham Green than the immediate selection treatment. Polymerase Chain Reaction (PCR) with specific npt IT oligonucleotide primers confirmed the presence of the selectable marker gene in all the 22 putative transgenic plants recovered. An inheritance study on the selfed progeny of transformed lines, confirmed transmission of the kanamycin resistance and pointed to a single insertion of the npt II gene in most of the transformed lettuce lines. The presence of the antibacterial genes was established in all the transformed plants, by PCR. The presence and effectiveness of the antibacterial peptides were determined by an in vitro assay and greenhouse evaluation of pathogen resistance in seedlings. Crude leaf extracts from selected transformed lines and control of untransformed Cobham Green were added to cultures of Erwinia carotovora subsp. carotovora and Xanthomonas campestris pv vitians to establish their effect on the growth of the pathogens. Growth of pathogens in cultures with crude extracts from transformed lines was inhibited while extracts from the control showed no such effect on the growth of the pathogens. In the greenhouse, selfed progeny of transformed lines and untransformed control were inoculated with the two lettuce pathogens to assess their response to disease development. Overall, delayed symptoms and reduced disease severity characterised the inoculated transformed lines, compared to the control seedlings. The potential of the antibacterial genes to effectively improve the resistance of bacterial diseases in lettuce was evident in these assays.