Protein self-assembly and protein-induced DNA morphologies

The ability of biomolecules to associate into various structural configurations has a substantial impact on human physiology. The synthesis of protein polypeptide chains using the information encoded by DNA is mediated through the use of regulatory proteins, known as transcription factors. Some transcription factors perform function by inducing local curvature in deoxyribonucleic acid (DNA) strands, the mechanisms of which are not entirely known. An important architectural protein, eleven zinc finger CTCF (11 ZF CTCF) is involved in genome organization and hypothesized to mediate DNA loop formation. Direct evidence for these CTCF-induced DNA loops has yet to be observed. In this thesis, the effect of 11 ZF CTCF on DNA morphology is examined using atomic force microscopy, a powerful technique for visualizing biomolecules with nanometer resolution. The presence of CTCF is revealed to induce a variety of morphologies deviating from the relaxed state of control DNA samples, including compact circular complexes, meshes, and networks. Images reveal quasi-circular DNA/CTCF complexes consistent with a single DNA molecule twice wrapped around the protein. The structures of DNA and proteins are highly important for operations in the cell. Structural irregularities may lead to a variety of issues, including more than twenty human pathologies resulting from aberrant protein misfolding into amyloid aggregates of elongated fibrils. Insulin deficiency and resistance characterizing type 2 diabetes often requires administration of insulin. Injectable and inhalable delivery methods have been documented to result in the deposition of amyloid fibrils. Oligomers, soluble multiprotein assemblies, are believed to play an important role in this process. Insulin aggregation under physiological conditions is not well understood and oligomers have not yet been fully characterized. In this thesis, in vitro insulin aggregation at acidic and neutral pH is explored using a variety of techniques, including kinetic thioflavin T fluorescence, circular dichroism spectroscopy, atomic force and electron microscopy imaging. The size distribution of insulin oligomers at different assembly stages is characterized through covalent cross-linking and gel electrophoresis. Results show that at the earliest assembly stage, oligomers comprise up to 40% and 70% of soluble insulin at acidic and neutral pH, respectively. While the highest oligomer order increases with insulin concentration at acidic pH, the opposite tendency is observed at neutral pH, with heptamers formed in 10[micrometre] insulin. These findings suggest that oligomers may be on- and off- pathway assemblies for insulin at acidic and neutral pH, respectively. Agitation, required to induce insulin aggregation at neutral pH, increases fibril formation rate and fibrillar mass by an order of magnitude each. Insulin incubated under agitated conditions at neutral pH rapidly aggregates into large micrometer-sized aggregates, which provides insight into injection-site amyloidosis and toxic pulmonary aggregates induced by administration of extraneous insulin.