Masters Theses

Date of Award

12-2009

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Albrecht von Arnim

Committee Members

Andreas Nebenführ, Elena Shpak, John Biggerstaff

Abstract

Bioluminescence is a phenomenon in which chemical energy is converted into light energy. Here, the oxidation of a luciferin substrate, catalyzed by a luciferase enzyme, results in the emission of a photon. This biological process is exploited in a technology referred to as Bioluminescence Resonance Energy Transfer (BRET). As its name implies, BRET depends on a nonradiative energy transfer event that occurs between a donor luciferase and an acceptor fluorophore. Fusion of the donor and acceptor molecules to a protein(s) of-interest allows one to identify and monitor molecular events, such as protein interactions or hormone binding events, based solely on the spectral properties of the light produced.

A primary goal of this research was to use BRET to investigate protein-protein interactions. Traditionally, BRET has been used to detect intermolecular interactions between protein pairs. To this end, BRET was applied to explore putative interactions between transcriptional regulators essential for organ polarity and floral development in Arabidopsis, FILAMENTOUS FLOWER (FIL), YABBY5 (YAB5), and LEUNIG (LUG). Results indicated that FIL and LUG are likely to interact in planta, supporting previous hypotheses that they function together within a protein complex.

BRET has also been utilized to identify intramolecular, conformational changes that occur following a ligand-protein binding event. Hormone-binding sensors, in particular, have seen considerable success and are now used to monitor changes in small molecule concentrations within a cellular context. To identify whether BRET-based sensors are applicable to plant hormone studies, three sensors were created, incorporating AUXIN BINDING PROTEIN 1 (ABP1), GIBBERELLIC ACID INSENSITIVE DWARF 1A (GID1A), and CHICKEN THYROID HORMONE RECEPTOR, LIGAND BINDING DOMAIN (cTRá-1 LBD). Data obtained indicated that with modifications, single-molecule BRET sensors may be suitable for use in plants.

Finally, luminescence imaging allows one to observe BRET as it occurs over a period of time. An imaging system was set up and used to monitor BRET occurring within transgenic Arabidopsis seedlings harboring either hRLUC-YFP or hRLUC constructs. Taken together, results from each of these studies highlight the versatility and sensitivity of BRET, and show that it can be used to monitor molecular events in a variety of applications.

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