Numerous biological phenomena occur as a result of macromolecular interactions. Metal-ion-biomolecule binding account for a large portion of these reactions, and unsurprisingly, a vast amount of new research in this area is constantly emerging. Gaining insight into the characteristics that define these interactions; including equilibrium fluctuations, metal center formation, global stability perturbation, cooperativity, allostery, and site-specific binding are all significant. As with all chemical reactions, biological interactions are regulated by thermodynamics; and the development of novel tools and methods by which to study these interactions becomes highly relevant. In this dissertation, three systems involving macromolecular binding are studied using well established biophysical techniques in conjunction with a critical look at appropriate uses for mathematical modeling. The first system studied is that of the serpin plasminogen activator inhibitor-1 (PAI-1). PAI-1 is a protease inhibitor that specifically effects fibrinolysis, or the process that prevents the formation of blood clots, and misregulation of this enzyme leads to uncontrollable hemorrhaging. ITC was utilized to investigate the thermodynamics of copper binding to PAI-1. Human carbonic anhydrase II (CA) was the second system investigated. Studies were conducted on zinc(II) and copper(II) binding to CA, a metalloenzyme responsible for acid-base balances in the blood and the transport of carbon dioxide. Interestingly, CA binds two copper(II) ions, one at the active site, and one at a higher affinity N-terminal site. Temperature dependent ITC, CD and GdnHCl denaturation studies were performed to explore the impact of copper(II) binding, particularly at the higher affinity N-terminal site. Finally, protein binding to inorganic gold nanoparticles (AuNPs) was investigated. AuNPS are utilized in areas of diagnostics, biological sensing and drug delivery. We studied binding of nanoparticles to a set of six biologically relevant proteins; glutathione, wild-type GB3, K19C GB3 (a variant at position 19), bovine CA, bovine serum albumin, and fibrinogen. Nanoparticle-protein binding was monitored via UV-Visible extinction and polarized resonance synchronous spectroscopy (PRS2). The UV extinction maxima wavelength shifts were fit with two models, a Langmuir isotherm model and a mass action-derived model. The models fit the data equally well, however, they predict very different Kd values, specifically for smaller sized AuNPs.
| Identifer | oai:union.ndltd.org:MSSTATE/oai:scholarsjunction.msstate.edu:td-1899 |
| Date | 01 May 2020 |
| Creators | McConnell, Kayla Diane |
| Publisher | Scholars Junction |
| Source Sets | Mississippi State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
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