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Protein-Ligand Interactions: Discovering Potential Small-Molecule Therapies for Alzheimer's Disease and Investigating the Effect of Non-Natural Amino Acid Incorporation on Enzyme-Substrate Interactions

Wong, Hann-Chung
Format
Thesis/Dissertation; Online
Author
Wong, Hann-Chung
Advisor
Kwon, Inchan
Abstract
Protein-ligand interactions are pivotal and ubiquitous in biological systems. Ligands are small molecules that include therapeutics, substrates, cofactors, inhibitors and many other species that can either influence protein function or be acted upon by a protein. Our research efforts focus on two main areas of study. The first is discovery of potential therapies for treating Alzheimer’s disease, directed against amyloid-beta (Aβ) pathology, and the second is developing a better understanding of substrate-enzyme behavior after mutation with genetically encoded non-natural amino acids. Here we describe four works that study protein-ligand interactions. First, Brilliant Blue G (BBG) was found to be an effective Aβ aggregation modulator and reduces Aβ-associated neurotoxicity by promoting the formation of off-pathway, non-toxic aggregates. Structure-activity analysis between BBG and its three commercially-available analogues (Brilliant Blue R, Brilliant Blue FCF, and Fast Green FCF), revealed that of the group, BBG was the most effective modulator of Aβ aggregation and cytotoxicity, and that its additional methyl groups are important for its enhanced modulating activity. In a follow up study, BBG was also found to disaggregate Aβ40 fibrils. Second, we found that erythrosine B was able to modulate both Aβ aggregation and Aβ-associated neurotoxicity. Given that this molecule uniquely possesses heavy halogen groups, through structure-function analysis, we demonstrated that halogenation is responsible for the Aβ modulating activities of erythrosine B and its analogues. The specific types, arrangement and placement of the halogen substituents dictated binding attributes, and also the aggregation and cytotocity modulating activity towards Aβ. Analysis by a novel competitive-binding assay with sequence-specific antibodies revealed that erythrosine B and a number of analogues interact with Aβ by binding at its N-terminus. Third, we discovered that incorporation of 3-(2-naphthyl)-alanine (Nal) at Glu30 in the catalytic domain of murine dihydrofolate reductase (mDHFR) can alter the allosteric cooperativity of the enzyme. Comparison with the closest natural amino acid analogue mutation, Trp at position 30, showed that the behavior promoted by Nal results from its unique size and shape. This is the first report showing that an expanded set of genetically encoded amino acids can alter the cooperativity of an enzyme. Evaluation of enzyme titers also revealed that the altered allosteric cooperative behavior is beneficial to the E. coli expression host. Lastly, we investigated the effects on enzyme function of incorporating Nal, a non-natural amino acid, within the hydrophobic core and solvent-exposed sites of mDHFR. Hydrophobic-core mutants: Through mutational analysis, a computed measure of steric incompatibility was found to have statistically significant monotonic correlations with key kinetic parameters (Km, kcat and kcat/Km) of mDHFR. Despite being related structurally, Nal was not an equivalent substitution for Trp and Phe, as it not only caused qualitative secondary structure changes, but also adversely affected catalytic activity, however, without impacting substrate binding. Solvent-exposed-site mutants: Nal, a large hydrophobic residue, could be incorporated at solvent-exposed sites (Phe142 and Phe179) without any adverse affects on the binding (Km) and catalytic (kcat) components of mDHFR enzymatic function. Results also revealed that structural changes were related to changes in hydrophobicity changes caused by mutation. Statistical analysis revealed that substituting Nal for Glu caused significant deviations in Km when compared to substitution of Nal at hydrophobic residues by an average of +0.60 µM (Student’s t-test, p < 0.01). These results demonstrate that mutations that minimize impact to structure can be strategically used to minimize impact on enzyme function.
Language
English
Published
University of Virginia, Department of Chemical Engineering, PHD (Doctor of Philosophy), 2015
Published Date
2015-06-24
Degree
PHD (Doctor of Philosophy)
Collection
Libra ETD Repository
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