Characterization of Vitronectin and Plasminogen Activator Inhibitor Type 1: Insights into Metal Binding, and Production of Reagents to Facilitate Structural Studies

Author

Cynthia Lee Brown, University of Tennessee - Knoxville

Date of Award

12-2008

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Cynthia B. Peterson

Committee Members

Engin Serpersu, Nitin Jain

Abstract

Vitronectin is a multifunctional glycoprotein involved in regulation of such processes as hemostasis, cell migration, immunity, and tumor metastasis. Many of its functions require interaction with various binding partners. One such partner, plasminogen activator inhibitor-type 1 (PAI-1), belongs to the serine protease inhibitor (serpin) superfamily of proteins. PAI-1 helps regulate hemostasis by affecting blood clot breakdown, and helps regulate cell migration and tissue remodeling by affecting extracellular matrix digestion. Our laboratory is pursuing structural information on vitronectin and the complex formed between vitronectin and PAI-1. To this end, the work presented in this thesis focuses on two aims: characterizing the interaction of vitronectin and PAI-1 with metal ions, and developing reagents to be used in structural studies of vitronectin. We have used electron paramagnetic resonance (EPR) spectroscopy to study metal binding by vitronectin and PAI-1. We have tested vitronectin and PAI-1 for binding of Mn²⁺ and Cu²⁺, and also established the feasibility of studying protein-Cu²⁺ interaction in solution by EPR, using bovine serum albumin (BSA) and Cu²⁺. In another set of experiments, we have used activity assays to assess the effect of metal ions on PAI-1 activity and on stabilization of PAI-1 by vitronectin. Regarding reagents, we have expressed and purified active, perdeuterated PAI-1, which can be used in complex with vitronectin in neutron scattering and NMR experiments. We characterized several monoclonal antibodies to vitronectin, and identified one suitable for use in co-crystallization screens. These accomplishments will aid in development of a more detailed picture of the interaction between vitronectin and PAI-1.

Recommended Citation

Brown, Cynthia Lee, "Characterization of Vitronectin and Plasminogen Activator Inhibitor Type 1: Insights into Metal Binding, and Production of Reagents to Facilitate Structural Studies. " Master's Thesis, University of Tennessee, 2008.
https://trace.tennessee.edu/utk_gradthes/367