Characterization of a bifunctional globin coupled sensor protein
Open Access
- Author:
- Patterson, Dayna
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 01, 2021
- Committee Members:
- Timothy Mcnellis, Outside Unit & Field Member
Joseph Cotruvo, Major Field Member
Squire Booker, Major Field Member
Emily Weinert, Chair & Dissertation Advisor
Philip Bevilacqua, Program Head/Chair - Keywords:
- heme
bacterial signalling
gas sensing
biochemistry
c-di-GMP
pGpG - Abstract:
- Heme proteins are found within all domains of life and play many key roles, including in diatomic gas sensing. Despite the large body of research on heme proteins, the molecular basis for the ability of the heme protein to modulate heme electronics and undergo conformational changes in response to gas binding to or the oxidation of the heme is still poorly understood. This is particularly true for the recently discovered class of heme proteins, known as globin coupled sensor (GCS) proteins. While evolutionarily related to mammalian globins, GCS proteins are found primarily in bacteria and archaea and serve as gas sensors that regulate activity of output domains within the same polypeptide chain. GCS proteins are involved in regulating bacterial biofilm formation (bacterial communities that form on surfaces and are recalcitrant to antibiotics), motility, and virulence. This dissertation presents the biochemical and structural characterization of DcpG, a diguanylate cyclase phosphodiesterase GCS protein from the bacterium Paenibacillus dendritiformis C454. DcpG is a bifunctional cyclic dimeric guanosine monophosphate (c-di-GMP) metabolic protein with GGDEF (Glu-Glu-Asp-Glu-Phe) and EAL (Glu-Ala-Leu) domains (synthesize and hydrolyze c-di-GMP, respectively) that are regulated by an N-terminal heme-bound sensor globin. C-di-GMP metabolic proteins are of great interest due to the role of c-di-GMP in controlling important bacterial phenotypes such as biofilm formation, motility, and virulence. However, the regulation of bifunctional c-di-GMP metabolic proteins is very poorly understood, despite putative homologs in thousands of bacterial genomes. The goal of the dissertation is to understand which gaseous ligand(s) regulate DcpG bifunctional enzyme activity and to probe its signal transduction mechanism. This first section of this dissertation reports the initial characterization of DcpG. DcpG bifunctional enzyme activity is differentially regulated by oxygen (O2) and nitric oxide (NO) in which this work highlights the first example, to our knowledge, of ligand binding to a heme sensor domain causing differential regulation of output domain activity. In addition, DcpG possesses unusual heme properties that include irreversible high midpoint potentials and rapid O2 and NO dissociation rates from the heme. By combining small angle X-ray scattering and negative stain electron microscopy data, the first structural model of a full-length globin coupled sensor protein was generated. These studies demonstrate for the first time that diatomic ligands (O2, NO, CO) can differentially modulate enzymatic domain activity within one heme sensor protein and allow bacteria to use a single protein to sense multiple gases within the environment. In the second section of the dissertation, the signal transduction mechanism within DcpG is probed. Enzyme kinetics, resonance Raman spectroscopy, and structural techniques are used to identify roles for heme-edge amino acids in modulating heme ligand binding and O2 dependent signal transduction. These studies identify an unusual heme-edge histidine residue that controls O2 dissociation, enzyme kinetics, and protein conformation. The last section of the dissertation focuses on establishing a high-throughput method to determine c-di-GMP specific phosphodiesterase activity of EAL domain containing enzymes. In addition, this section of the dissertation highlights future and ongoing work of DcpG characterization to further understand its structure and its role in vivo. In summary, this work has expanded our knowledge of GCS proteins and bifunctional c-di-GMP metabolic enzymes. This work suggest that DcpG may play a role in Paenibacillus dendritiformis C454 behavior in response to its gaseous environment. In addition, DcpG unusual heme properties play a role in its signal transduction mechanism, from gas sensing to bifunctional enzyme activation. Overall, this dissertation provides a foundation to understand of c-di-GMP enzymes that are activated by gaseous ligands.