Application and Optimization of Bioluminescence Resonance Energy Transfer (BRET) for Real Time Detection of Protein-Protein Interactions in Transgenic Arabidopsis as well as Structure-Based Functional Studies on the Active Site of Coelenterazine-dependent Luciferase from Renilla and its Improvement by Protein Engineering
Date of Award
Doctor of Philosophy
Albrecht von Arnim
Beth Mullin, Andreas Nebenfuhr, Dan Roberts, Liz Howell
Bioluminescence resonance energy transfer (BRET) is a biological phenomenon in some marine organisms such as Renilla reniformis and Aequorea victoria. In BRET, resonance energy from decarboxylation of coelenterazine, a substrate of Renilla luciferase (RLUC), is transferred to its acceptor such as green fluorescent protein (GFP) or yellow fluorescent protein (YFP), dependent on a distance of around 5 nm between the energy donor (RLUC) and its acceptor. The activation of the energy acceptor results in a spectral change in luminescence emission. The BRET system allows investigation of in vivo protein-protein interactions in real time. This was demonstrated with two heterodimeric interactions in transgenic Arabidopsis.
In an attempt to optimize the activity and to address the reaction mechanism of the RLUC enzyme, a homology model of RLUC was obtained using a haloalkane dehalogenase, LinB, as a template. Furthermore, the homology model and the crystal structures of RLUC were docked with coelenterazine. The computational analyses suggested potential roles of catalytic triad residues (Asp120, Glu144, and His285) and substrate binding residues (N53, W121, and P220) in the active site. Mutagenesis, spectroscopy, and expression in E. coli were carried out to elucidate the reaction mechanism of RLUC and the possible roles of the residues. Moreover, the catalytic triad was probed using pharmacological tests. Using random mutagenesis, a new triple mutant was isolated, which showed increased kcat, increased half-life, and higher resistance to substrate inhibition. These results establish enzymatic characteristics of RLUC and, furthermore, suggest that the triple mutant may result in potentially advantageous properties for BRET assays, including imaging routines in Arabidopsis.
Woo, Jongchan, "Application and Optimization of Bioluminescence Resonance Energy Transfer (BRET) for Real Time Detection of Protein-Protein Interactions in Transgenic Arabidopsis as well as Structure-Based Functional Studies on the Active Site of Coelenterazine-dependent Luciferase from Renilla and its Improvement by Protein Engineering. " PhD diss., University of Tennessee, 2008.