Elucidating the Various Roles of the Globin Domain from Globin Coupled Sensors 公开
Rivera, Shannon (Spring 2019)
Abstract
Globin coupled sensors (GCS) are sensory proteins used by bacteria to determine the surrounding gaseous environment. The function of a GCS is determined by the output domain of the GCS, which include phosphodiesterases, kinases, and diguanylate cyclases (DGC). Diguanylate cyclase domains produce cyclic dimeric guanosine monophosphate (c-di-GMP) from guanosine triphosphate (GTP). C-di-GMP is a bacterial secondary messenger and a major regulator of biofilm formation. Pectobacterium carotovorum ssp. carotovorum and Bordetella pertussis both contain GCS proteins (PccGCS and BpeGReg, respectively) with DGC output domains. Previous works has shown that oxygen binding in the globin domain regulates the output domain, but the signaling mechanism and structure of GCSs are not well characterized.
Here we present our work on elucidating these previously uncharacterized properties. In doing so, the isolated globin domains from PccGCS (PccGlobin) and BpeGReg (BpeGlobin) have been characterized. The oligomeric state of PccGlobin is dimeric while BpeGlobin is monomeric, indicating potential oligomer binding sites in the globin domain. As full length PccGCS and BpeGReg exist as different oligomeric states (dimer-tetramer-octamer and monomer-dimer-tetramer, respectively), the globin domain appears to be a major determinant of oligomerization. Additionally, the globin truncations revealed altered oxygen dissociation kinetics, as compared to PccGCS and BpeGReg.
Furthermore, crystallization of BpeGlobin allowed for additional identification of key residues in the heme pocket. Site-directed mutagenesis has been used to interrogate the relative roles of each of these residues in stabilizing bound O2 and contributing to each dissociation rate in both the full-length and isolated globin proteins. Further investigation into the heme pocket and the dimerization of the globin domain has been accomplished using domain swapping, cross-linking, Fourier Transform Infrared (FTIR) spectroscopy, electrochemistry and more. This work elucidates the global effects of protein oligomerization on conformation of the globin domain, as well as identifies key requirement for signal transduction within the globin coupled sensor family.
Table of Contents
Contents
Chapter 1: Introduction to Globin Coupled Sensor Signaling
1. Introduction 2
2. General Structure and Ligand Binding Characteristics 4
3. HemAT-Bs: The First GCS Characterized 7
4. Diguanylate Cyclase-Containing GCS Proteins 11
5. Stressosome-Related GCS Proteins 24
6. Conclusion 27
7. Chapter References 28
Chapter 2: Characterizing GCS Globin Domain Interactions
1. Introduction 37
2. Experimental Results and Discussion 40
3. Conclusion 51
4. Chapter References 52
Chapter 3: Structural Insights into Globin Coupled Sensors
1. Introduction 58
2. Experimental Results and Discussion 61
3. Conclusion 77
4. Chapter References 78
Chapter 4: Determining Heme Residue Effects on Kinetic Turnover in GCSs
1. Introduction 85
2. Experimental Results and Discussion 88
3. Conclusion 96
4. Chapter References 97
Chapter 5: Conclusion - Signaling in Globin Coupled Sensors Proteins
1. Introduction 103
2. Knowledge Gained 103
3. Proposed Future Work 105
4. Chapter References 106
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