Dissertations, Theses, and Capstone Projects

Date of Degree

2-2024

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Kevin H. Gardner

Committee Members

Jean P. Gaffney

Mark Emerson

Stefan U. Pukatzki

Jared E. Toettcher

Subject Categories

Biology | Biotechnology | Cell Biology | Structural Biology

Keywords

Saccharomyces cerevisiae, Flavin Mononucleotide, EL222, BcLOV4, Epigenetics

Abstract

A variety of cellular biosensors have evolved which can translate electromagnetic radiation from the environment into biological functions. Light-Oxygen-Voltage (LOV) domains in particular, are a group of photoreceptors that respond to blue wavelengths of light and can become a cellular photoswitch by converting this stimulus through their attached effector domains. For instance, the Helix-Turn-Helix (HTH) DNA-binding activity of the bacterial transcription factor EL222 and the Regulator of G-protein Signaling (RGS) in the fungal membrane associating protein BcLOV4 are both induced by blue-light sensing LOV domains. While previous work has provided the field with a structural characterization of LOV proteins in the dark state, the challenge has been to fully characterize the structural properties and mechanisms of action in the functional lit states. Owing to the highly dynamic conformation-state changes driven by blue-light activation, the structure-function relationship of these LOV proteins in the light-activated state remains elusive. This doctoral thesis employs a combination of biological and biochemical methods to derive novel insight to the mechanistic and structural properties of both EL222 and BcLOV4, which in turn has aided the engineering of new light-based tools for controlling cellular biology.

In Chapter 2 of my thesis, I performed mechanistic studies to develop an early model of functional activity in EL222 including site-directed mutagenesis, in vivo fluorescence characterization, and kinetics in conjunction with in vitro classifications. Using flow cytometry-based transcriptional activity assays of 23 EL222 mutants in the budding yeast Saccharomyces cerevisiae produced a dynamic range of expression profiles. A group of functionally inactive mutants, most notably EL222R215A, revealed residues critical to homodimer-complex formation and DNA binding. Surprisingly, a mechanistically important EL222D212A mutation drove allosteric like effects on ligand binding affinity resulting in dark state activity, providing the first evidence of EL222 acting as a hybrid sensor (ligand/light). These data, in combination with in vitro structural classifications, provide the most accurate, empirically driven structure-function model of light-activated EL222 to date.

BcLOV4 is a fungal LOV protein in the Regulator of G-protein Signaling family (RGS-LOV) that undergoes light-induced translocation to the plasma membrane and is reversible in the dark. No structure of this protein is available, and the mechanistic properties and kinetics of membrane association/dissociation are poorly understood. In Chapter 3, I employed confocal microscopy and analysis of fluorescently tagged BcLOV4 in yeast to classify over 30 targeted residue mutations influencing membrane association/dissociation kinetics in vivo. Both alanine and glutamic acid mutations at the Ja helix/LOV b-sheet interface resulted in an ~4-fold increase in membrane dissociation kinetics. Contrary to our expectations, when we investigated the photochemical kinetics of LOV chromophore, we observed a slower photocycle indicating this is not coupled to membrane release. Our data aided the collaborative production of an experimentally driven HADDOCK model fitted to a low resolution cryo-EM map producing the first structural basis of photoactivation of an RGS-LOV protein.

There is a great demand for research and development of novel tools and biosensors to probe complex biological questions in vivo. Successful development of LOV-based optogenetic tools include prerequisites for high spatiotemporal control, sufficient signal to noise, and low toxicity to host processes. In Chapter 4, I designed and engineered a novel light-activated optogenetic tool using EL222 fusion proteins and performed proof of concept experiments on light-driven chromatin modifications and gene expression. Using transcriptional assays, we tested the translocation properties of EL222-fusion proteins (either the histone acetyltransferase enzyme GCN5, Acetyl-CoA synthetase ACS2, or transcription factor PHO4) to the gene promoter region of the endogenous yeast acid phosphatase PHO5 and observed successful gene activation in some cases. These preliminary data provide proof of concept for a one component light-induced system employing EL222 fusion proteins. Together, my thesis provides novel insight to the structure-function relationship of two LOV proteins with unique properties and promotes future studies to address their endogenous mechanistic roles. The structural characterization of light induction in the LOV class photoreceptor and early insight gleaned from my development of an EL222-based optogenetic tool provides an avenue for the continual improvement of innovative research tools to probe biological questions in a variety of systems.

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