Dissertations, Theses, and Capstone Projects

Date of Degree

9-2021

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Kevin Gardner

Committee Members

David Jeruzalmi

Zimei Bu

Prabodhika Mallikaratchy

Brian Crane

Subject Categories

Biochemistry | Biochemistry, Biophysics, and Structural Biology | Biophysics | Molecular Biology | Other Biochemistry, Biophysics, and Structural Biology | Structural Biology

Keywords

EL222, Mechanism, Structure, HDX-MS, Trancription factor, Blue light photoreceptors, signal transduction, optogenetics

Abstract

Photoreceptors play a crucial role in signal transduction as specialized proteins which sense light as environmental stimuli and transduce the signal to control of downstream functions. Here we focus our attention on one class of these proteins, the Light-Oxygen-Voltage (LOV) domain, which is sensitive to blue light via an internally-bound flavin chromophore. Since their initial discovery in plant phototropins, many details of their photochemistry, chromophore interactions, and use with a diverse set of functional effectors have been described. However, several key details, especially a comprehensive understanding of signaling mechanism and its regulation, still remain elusive due in part to the challenges trying to characterize the highly dynamic active states of full-length LOV proteins. Here, we focus on filling this gap in knowledge by determining the structures of active states and investigating the dynamics of these EL222 LOV-Helix-Turn-Helix (HTH) transcription factor as a test case. Upon illumination, photochemical changes at an internally-bound FMN chromophore triggers intra- and inter-domain rearrangements leading to release of the HTH domain from a dark-state inhibited state, allowing EL222 to dimerize and bind DNA. Starting with a known dark state crystal structure and a data from a variety of biophysical approaches, including EPR, NMR and HDX-MS, to model EL222 active states. Furthermore, we probed into inherent dynamics of these systems using site-directed mutagenesis of intra- and inter-protein interfacial residues to elucidate the importance of these sites in EL222 regulation. We also uncover an unexpected role of FMN occupancy in EL222 activity, providing a second way to control DNA binding. Overall, in this study we present a light-activated light state structure model and shed some light on the mechanistic insights into regulation by two different types of stimuli. In addition, we provide information on the energetic importance of several key residues in photosensing by this protein, with implications in engineering new optogenetic systems or fine-tuning these exciting systems.

Download

Share

COinS