Elucidating the Various Roles of the Globin Domain from Globin Coupled Sensors Pubblico

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    

About this Dissertation

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Chemistry
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Chemistry, Physical Chemistry, Inorganic
Parola chiave	Globin Coupled Sensor Signaling Pathway Protein
Committee Chair / Thesis Advisor	Dr. Emily Weinert, Emory University
Committee Members	Dr. Stefan Lutz, Emory University Dr. Brian Dyer, Emory University

Ultima modifica

Primary PDF

Thumbnail	Title	Date Uploaded	Actions
	Elucidating the Various Roles of the Globin Domain from Globin Coupled Sensors ()	2019-04-01 16:25:20 -0400	Press to Select an action Download

Supplemental Files

Thumbnail	Title	Date Uploaded	Actions