Gene mutation and biochemical characterization of recombinant mutant FtrB: A novel cupredoxin

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Jessica D Buchanan (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: Brucella is a Gram-negative zoonotic pathogen that is commonly passed from livestock to humans. The resulting condition, Brucellosis, is hard to diagnose, hard to treat, and chronic. Brucella is able to overcome the toxic environment of the host cell by utilizing various transport systems that steal essential nutrients, such as iron, from the host. The FtrABCD system of Brucella is an example of one of these transport systems involved in Fe2+ uptake and contributes to the high virulence of this bacteria. Periplasmic FtrB is hypothesized to be novel cupredoxin due to its common ancestry with known cupredoxins with a conserved Type-1 Cu binding site. However, FtrB does not conserve two His and a Cys Type-1 Cu coordinating residues seen in other cupredoxins. Previous homology modeling of wild-type Brucella FtrB shows Asp, Met, and His residues forming a putative Cu binding pocket. To test the proposed metal binding of these residues, single-site substitution mutants of FtrB (D55A, M81A, and H121A) were created. Isothermal Titration Calorimetry (ITC) results indicated greater binding affinity in the mutants than the wild-type protein. Circular Dichronic (CD) spectroscopy suggested significant structural differences for both mutants with H121A having more pronounced structural changes. The purpose of this study was to confirm the binding residues identified in the homology model bound Cu and mutations of these sites would eliminate Cu binding. Characterization assays performed on these mutants indicate structural and binding differences when compared to the wild-type.

Additional Information

Publication

Thesis

Language: English

Date: 2023

Subjects

mutagenesis;isothermal titration calorimetry;circular dichroism;cupredoxin

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