Protein-DNA Affinity Binding Studies Using Mass Spectrometry and Optical Spectroscopy

The insulin-linked polymorphic region (ILPR) of the human insulin gene contains tandem repeats of similar G-rich sequences, some of which form intramolecular G-quadruplex structures in vitro. Previous work showed affinity binding of insulin to an intramolecular G-quadruplex formed by ILPR variant a, which is the predominant sequence. The present work expands the scope of the research to include interactions of insulin and the highly homologous insulin-like growth factor-2 (IGF-2) with 2-repeat sequences of ILPR variants a, h, and i (ILPRa, ILPRh and ILPRi, respectively). Circular dichroism spectroscopy (CD) indicates that ILPRa and ILPRh form intramolecular G-quadruplexes that are not observed for ILPRi. Affinity MALDI MS was used to compare capture of the proteins by the variants that were immobilized at MALDI probe surfaces and Surface Plasmon Resonance (SPR) was used to determine binding constants and kinetics of the interactions. Insulin and IGF-2 exhibited high binding affinity for ILPRa and ILPRh but not ILPRi, indicating the involvement of intramolecular G-quadruplexes. A particularly interesting result was the observation of a dramatic increase in binding strength, but only for insulin with ILPRa, when insulin concentration was decreased below nM. This result suggests that insulin may have more than one mode of interaction with ILPRa. Studies were then performed to see if complexes formed between ILPR oligonucleotides ILPRh and ILPRi and the proteins could be detected by MALDI MS. The use of covalent cross-linking to preserve the complexes was investigated. Although the cross-linking experiments were not successful, it was possible to detect significant complexation of both insulin and IGF-2 with ILPRh but not ILPRi. The intramolecular G-quadruplex structure of ILPRh is the likely source of the interactions since it is absent in ILPRi. Two different methods were used to determine the location of the binding sites in insulin and IGF-2 for the G-quadruplexes formed by ILPRa and ILPRh. Both methods employed affinity capture of peptides produced by enzymatic digestion of insulin and IGF-2. The sequence VCG(N)RGF was found to be common to all of the insulin and IGF-2 peptides captured by ILPRa and ILPRh, with the exception of those peptides that contained disulfide bonds and those peptides captured by both ILPRh and ILPRi in the affinity MALDI MS experiments. The common sequence is believed to play a key role in the affinity binding of insulin and IGF-2 with the G-quadruplexes formed by ILPRa and ILPRh. Results also showed the importance of arginine in the common sequence in the binding interactions. Knowledge of the affinity binding site in insulin and IGF-2 for G-quadruplexes formed by the ILPR oligonucleotides offers new insight into these interactions that could be used to design affinity binding reagents as well as increasing our understanding of the specificity of G-quadruplex-protein interactions. The last part of this dissertation describes the investigation of Indium Tin Oxide (ITO)-coated glass slides instead of fused silica plates that were used up to this point in the research as the substrate for immobilization of ILPR oligonucleotides for affinity MALDI MS of proteins. ITO-coated glass slides were shown to provide better signal reproducibility, peak resolution, detectability and hydrophobicity compared to the fused silica plates. The results of the protein capture studies showed that protein detectability was improved by two orders of magnitude for insulin and one order of magnitude for IGF-2. Capture of the insulin beta-chain from DTT-treated commercial human serum that was diluted 104-fold in order to reduce...