The Mechanistic Studies of Dinoflagellate Bioluminescence and the Conformational Analysis of Dinoflagellate Luciferase

Abstract

This dissertation discusses the luciferase enzyme of dinoflagellates, which are eukaryotic microorganisms found in both freshwater and marine environments. Dinoflagellates are known for their capability for bioluminescence. Dinoflagellate bioluminescence is produced upon physical agitation, triggering the oxidation of an open-chain tetrapyrrole substrate commonly known as luciferin (LH2), with molecular oxygen which is mediated by the luciferase (LCF) enzyme. The enzyme is regulated by pH, active at acidic levels and inactive at basic levels. The change in pH is brought upon consequently though a signal transduction cascade subsequently from mechanical stimulation. Chapter two details the construction of LCF domain constructs and the application of fluorescence spectroscopy to analyze the pH-dependent conformational state and domain cooperative effects of LCF. Additionally, the Förster/Fluorescence Resonance Energy Transfer (FRET) based assay is introduced as an effective technique to measure protein conformational changes. Finally, the results of the fluorescence spectroscopy studies are discussed, which includes the successful construction of a plasmid encoding SUMO-tagged LCF-D3, the removal of DnaK contamination, and the purification of LCF constructs. Chapter three focuses on the kinetic investigation of dinoflagellate luciferase catalysis. Computational simulations utilizing constant pH accelerated molecular dynamics (CpHAMD) identified several amino acid residues possibly involved in the catalytic mechanism of LCF. Through mutagenesis, the roles of these active site residues were investigated also utilizing kinetic assays. Microfluidics-based assay was carried out to address challenges associated with the rapid kinetics of the wild type LCF-D3 reaction and the limited availability of oxygen-sensitive LH2. The results of the kinetic assays are discussed, detailing a single and double exponential decay models for LCF-D3 and LCF-D2 respectively, results of the LCF-D3 mutants, and the results of the microfluidics-based assay. Chapter four aims to gain insight into the structure and function of the unique luciferase of Noctiluca scintillans (Ns-LCF) using bioinformatic tools. Compared to the canonical LCF, rather than three homologues domains, Ns-LCF possess two domains, an LCF-like region and a luciferin binding protein (LBP)-like region. Finally, chapter five details other projects and the future direction of this dissertation.