The polyelectrolyte and the hydrophobic effects influence affinity of protein-DNA interactions. Explorations of these phenomena form the focus of this thesis as applied to three biological systems: lactose (LacI) and purine (PurR) repressors from bacterial cells and p53 protein from human cells.
First, the presence of guanine or DNA enhanced DNA or guanine binding by PurR, respectively. Compared to the behavior of LacI, a homologous protein, PurR exhibits tighter control of the pur regulon, an effect that derives from both thermodynamic and kinetic differences. In vivo lactose utilization---controlled by LacI---is response to an environmental opportunity to which cells need to respond rapidly. In contrast, purine enzyme biosynthesis---controlled by PurR---is an energetically costly process so that cellular response must integrate signals of purine states over a period of time.
Second, the ionic and temperature dependence of DNA binding---reflecting the polyelectrolyte and hydrophobic effects, respectively---for the structurally similar PurR and LacI proteins was examined. Both proteins exhibited a roughly ∼2-fold increase in ion pairs formed in their high affinity form. Both proteins employ basic residues from their N-terminal helix-turn-helix DNA binding motif and core domain; however, PurR employs additional basic residues from its hinge helices and possibly interchain N-terminal to core ion pairs to account for the ∼15 ion pairs when PurR-guanine complexes DNA. The values of Delta Cp determined by van't Hoff analysis, showed greater participation of local folding for PurR than LacI possibly due to more extensive hinge helix folding. These major differences between PurR and LacI, demonstrate that structurally similar proteins can display highly distinct behaviors.
Finally, ion concentration dependence of wild type, S392E, and a C-terminal deletion variant (Delta33) p53 proteins showed a remarkably low number of ion pairs formed compared to the number predicted from p53 core domain structural data. CD, temperature dependence, and water release studies of Delta33 p53 are consistent with the notion of a structurally rigid core DNA binding domain. However, the DNA binding properties of Delta33 p53 variant did not substantiate its characterization as "activated" form of p53 and disfavor the popular model of DNA binding regulation by the p53 C-terminal domain.
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/18596 |
| Date | January 2003 |
| Creators | Moraitis, Markos I. |
| Contributors | Matthews, Kathleen S. |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | 185 p., application/pdf |
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