GSBS Dissertations and Theses

ORCID ID

0000-0002-0560-2428

Publication Date

2020-01-29

Document Type

Doctoral Dissertation

Academic Program

Biochemistry and Molecular Pharmacology

Department

Biochemistry and Molecular Pharmacology

First Thesis Advisor

Stephen Miller

Keywords

bioluminescence, in vivo imaging, bioluminescence imaging, fatty acyl-CoA synthetase, evolution, enzymatic promiscuity, fatty acid amide hydrolase, fatty acid, luciferase, luciferin, structural biology

Abstract

Bioluminescence is the chemical production of light that results when a luciferase enzyme catalyzes the luminogenic oxidation of a small-molecule luciferin substrate. The numerous luciferases and luciferins nature has evolved can be used to illuminate biological processes, from in vitro assays to imaging processes in live animals. However, we can improve the utility of bioluminescence through modification of these enzymes and substrates. My thesis work focuses on developing reporters that expand the bioluminescent toolkit and improving our understanding of how bioluminescence works on a molecular level.

The first part of my thesis focuses on characterizing luciferases and luciferins that improve bioluminescence imaging in vivo. Some of our luciferins can outperform the natural D-luciferin substrate in live mouse imaging, while others are selectively utilized by mutant luciferases in live mouse brain. We also engineered luciferins that can selectively report on endogenous enzymatic activity in live mice.

The second part of my thesis focuses on determining the molecular details of how enzymes related to firefly luciferase, long-chain fatty acyl-CoA synthetases (ACSLs), can function as latent luciferases. I have determined the structure for one of these enzymes and improved its bioluminescent activity with synthetic luciferins enough to image in live mouse brain. I also characterized the selectivity in chimerized enzymes that combine firefly luciferase and ACSLs. In summary, my work improves the utility of bioluminescence for in vivo use and informs us about how evolutionarily-related enzymes function as luciferases on a molecular level.

DOI

10.13028/rhh8-xe30

Rights and Permissions

Licensed under a Creative Commons license

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.

Available for download on Thursday, March 04, 2021

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