Date of Award

December 2014

Degree Type

Thesis

Degree Name

Master of Science

Department

Physics

First Advisor

Valerica Raicu

Committee Members

Marius Schmidt, Dilano K. Saldin, Bimal Sarma

Keywords

Forster Resonance Energy Transfer, FRET, Micro-spectroscopy, Optimis, Ste2, Sterile-2 Receptor

Abstract

G-protein-coupled receptors (GPCRs) are the largest family of receptors that respond to a wide variety of extracellular stimuli, including molecular ligands such as odorants, neurotransmitters, and hormones, as well as physical agents sigh as light and pressure. The stimulation event results in initiating conformational changes in the structure of the receptor, which further results in the release of the heterotrimeric G-protein; the latter has a variety of functions within signaling pathways in cellular biology. The GPCR explored in this investigation is the Sterile 2 α-factor receptor (Ste2), whose natural function is that of a yeast mating pheromone receptor. Its natural ligand is the α-factor mating pheromone, and was used to study the interaction of dynamic hetero-oligomers of Ste2 receptors in the presence and absence of the ligand. Förster Resonance Energy Transfer (FRET), a non-radiative process of energy transfer between fluorescent molecules, was used to probe interactions between protomers within homo-oligomers of Ste2 in living Saccharomyces cerevisiae (yeast) cells and hence the general association stoichiometry and quaternary structure. Through the use of spectrally-resolved two-photon microscopy with pixel-level resolution, the interaction between differently fluorescent tagged proteins was explored by determining their apparent FRET efficiency at each pixel. It was found that Ste2 forms both dimers and tetramers, and with the introduction of its natural ligand, the equilibrium might be shifted from dimers to tetramers.

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