Date of Completion

4-29-2016

Embargo Period

10-25-2016

Keywords

ITC, NMR, Shc, BTN3A1, T Cell, Protein, Interaction

Major Advisor

Olga Vinogradova

Associate Advisor

Debra Kendall

Associate Advisor

Andrew Wiemer

Associate Advisor

Andrei Alexandrescu

Field of Study

Pharmaceutical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Proteins are crucial in virtually all cell activities. The vast majority of proteins interact with other molecules to function properly. The investigation of biomolecular interactions calls for structural and thermodynamic information acquired by a variety of experimental techniques and analytical methods. This dissertation is mainly concentrated on two frequently used and powerful tools, Isothermal Titration Calorimetry (ITC) and Nuclear Magnetic Resonance (NMR) spectroscopy, to investigate protein interactions. p52 Shc, a well-studied cellular adaptor protein, is a positive regulator in the mitogen-activated protein kinases pathway. We have confirmed that Shc interacts with both, integrin tyrosine(s)-phosphorylated cytoplasmic tails and phosphotidylinositol lipids, through the same Phospho-Tyrosine Binding (PTB) domain. The binding to phosphorylated peptides is enthalpy driven and tighter, while Shc interactions with PtdIns are entropy driven and much weaker. Shc interactions with PtdIns and β3-derived peptides are only weakly competitive with partially overlapping bindings sites. We also observed thermodynamic indications for potential intramolecular interactions within Shc, which could be perturbed by the binding to the phosphorylated receptor. BTN3A1, an immunoglobulin superfamily member, was found recently to be critical in Vγ9Vδ2 T cell activation. We described a phosphoantigen prodrug approach with great potency in Vγ9Vδ2 T cell stimulation. Our NMR and ITC results revealed the intracellular binding of BTN3A1 to phosphoantigens. In addition, we have provided biophysical evidence indicating the involvement of membrane proximal region in phosphoantigen binding and potential structural changes within BTN3A1 intracellular region. By focusing on p52 Shc and BTN3A1, I hope to demonstrate how a combination of structural and thermodynamic knowledge can be utilized to answer many important questions in signaling transduction close to the cell membrane.

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