Elucidating the Structure and Function of Heme Pocket Residues in Bacterial Diguanylate Cyclase-Containing Globin-Coupled Sensors Open Access

Abstract

The ability to respond and adapt to the environment is a hallmark of biological systems. Globin-coupled sensors are one method bacteria have evolved to respond to the important environmental stimulus, molecular oxygen (O2). To investigate the structure and signaling mechanisms of these proteins, the crystal structure of the globin domain from the diguanylate cyclase-containing globin-coupled sensor (DGC-GCS), BpeGReg, has been solved in its O2-bound state, making this the first bacterial sensor globin to be characterized in its native active state. Furthermore, key information about the role of heme pocket residues in ligand interaction has been obtained through Fourier-transform infrared spectroscopy (FTIR) on CO-bound BpeGReg and the closely related PccGCS, which identified a tyrosine residue in the heme distal pocket as the primary hydrogen bond donor in both proteins. Additionally, FTIR data revealed an ordered water molecule within the heme pocket of BpeGReg as an additional, previously unrecognized ligand interacting partner. Finally, the proximal histidine residue was identified as a major regulator of protein oligomeric state, as well as a contributor to ligand stability. These findings highlight the roles of heme pocket residues in modulating overall DGC-GCS structure and function.

Table of Contents

Introduction__________________________________________________________________ 1

    c-di-GMP Production and Regulation____________________________________________ 2

    Globin-Coupled Sensors_______________________________________________________ 5

    PccGCS and BpeGReg________________________________________________________ 9

Methods____________________________________________________________________ 10

    Protein Expression__________________________________________________________ 11

    Protein Purification__________________________________________________________ 11

    BpeGlobin Crystallization_____________________________________________________ 12

    FTIR Characterization_______________________________________________________ 12

    H2¹⁸O Buffer Exchange_______________________________________________________ 13

    Ultraviolet/Visible Spectroscopy_______________________________________________ 13

    High-Performance Liquid Chromatography_______________________________________ 14

Results_____________________________________________________________________ 14

    BpeGlobin Crystal Structure___________________________________________________ 14

    Heme Distortion____________________________________________________________ 20

    Fourier-Transform Infrared Spectroscopy________________________________________ 20

    Examination of Ordered Water_________________________________________________ 23

    Ultraviolet/Visible Spectroscopy_______________________________________________ 25

    High-Performance Liquid Chromatography_______________________________________ 26

Discussion___________________________________________________________________ 27

    Structural Insights___________________________________________________________ 27

    Distal Interactions___________________________________________________________ 31

    Roles of the Proximal Histidine________________________________________________ 33

Conclusions_________________________________________________________________ 36

References__________________________________________________________________ 37

Supporting Information_______________________________________________________ 39

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry Biology, Molecular
Keyword	Globin coupled sensors, diguanylate cyclase, heme protein, crystallography, FTIR, HPLC
Committee Chair / Thesis Advisor	Weinert, Emily, Emory University
Committee Members	Quave, Cassandra, Emory University Soria, José, Emory University

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