Dissertations, Theses, and Capstone Projects

Date of Degree

9-2021

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Kevin H. Gardner

Committee Members

Mark Emerson

Ruth Stark

Jayne Raper

Brian Chow

Subject Categories

Amino Acids, Peptides, and Proteins | Biochemistry | Bioinformatics | Biophysics | Cell Biology | Enzymes and Coenzymes | Integrative Biology | Lipids | Molecular Biology | Other Biochemistry, Biophysics, and Structural Biology | Structural Biology

Abstract

Light provides organisms with energy and spatiotemporal information. To survive and adapt, organisms have developed the ability to sense light to drive biochemical effects that underlie vision, entrainment of circadian rhythm, stress response, virulence, and many other important molecularly driven responses. Blue-light sensing Light-Oxygen-Voltage (LOV) domains are ubiquitous across multiple kingdoms of life and modulate various physiological events via diverse effector domains. Using a small molecule flavin chromophore, the LOV domain undergoes light-dependent structural changes leading to activation or repression of these catalytic and non-catalytic effectors. In silico analyses of high-throughput genomic sequencing data has led to the marked expansion in the collection of known LOV proteins. Many of these proteins contain new LOV-effector pairings, each of which can be predicted to have novel light-controlled function. Of these combinations, RGS (regulator of G protein signaling)-LOV proteins are an exciting new system for light regulation of cellular process and provide an interesting test case to study. Successfully establishing the structural role of LOV photoreceptors with RGS effectors will provide insights into LOV photoreceptor biochemistry that will both enhance understanding of the generality of LOV signaling and yield new optogenetic tools for manipulating biological circuitry.

In this dissertation, I build on a previous report in collaboration with Brian Chow’s group (detailed in Chapter 2) of RGS-LOV proteins utilizing a light-regulated and reversible electrostatic interaction between a polybasic amphipathic helix in the linker between the LOV and DUF (Domain of Unknown Function) domains to mediate binding to anionic plasma membrane phospholipids. With a combination of biophysical and biochemical methods, including cryo-EM, limited proteolysis, and HDX-MS, I report the first structural characterization of bcLOV4, a Botrytis cinerea RGS-LOV photoreceptor (Chapter 3). This study establishes the three-dimensional domain arrangement between the RGS, LOV, and DUF domains and gives insights into the domain-level light-triggered signal propagation, membrane recruitment, and function activation as a GTPase accelerating protein (GAP). To reveal more information about the well-conserved DUF domain that flanks the LOV at the C-terminus, I have tried both reductionist approaches as well as comparative analysis (outlined in Chapter 4). I characterized five different RGS-LOVs using bioinformatic techniques followed by heterologous overexpression in bacteria, purification, and initial solution evaluation. For the first time, I describe apRGS and ffRGS, which to date have not yet been experimentally characterized. This comparative study will aid in understanding general trends of the RGS-LOV class of proteins and give insights into their role in fungal photobiology.

In this work, I showed a potent combination of results here to help enhance our understanding on the RGS-LOV class and the role that the DUF plays in the overall structure-function. This work has substantial implications on understanding the ways that such proteins might be regulated in natural settings for control of GPCR signaling and further, for the engineering of RGS-LOVs as valuable components for creating optogenetic tools to perturb cellular signaling and physiology.

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