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Date of Award
Doctor of Philosophy (PhD)
Michael J. Knapp
Successful iron acquisition plays a crucial role in bacterial virulence. Pathogenic Gram-negative bacteriaPseudomonas aeruginosa have developed sophisticated hemeuptake systems to steal iron from hosts. One of these systems involves a cell surface heme receptor (PhuR), a periplasmic heme transport protein (PhuT), an inner membrane heme permease (PhuV) coupled with an ATPase (PhuU) and an associated protein (PhuW), and a cytosolic heme chaperone (PhuS).
In this study, phuT gene was cloned, over-expressed in E. coli and purified as a 33 kDa His-tagged protein with ~ 50% heme-loaded. Heme-free (apo) PhuT binds heme and protoporphyrin IX at 1:1 ratio with high affinity (Kd~ 1.2 and 14 nM, respectively). EPR, MCD, and UV-vis spectroscopies indicate a penta-coordinate ferric high-spin heme center with a low redox potential (below -300 mV/NHE) in PhuT. A well-ordered PhuT structure was observed upon heme binding via fluorescence and CD spectroscopies, which was then confirmed by limited proteolysis assays. A BLASTP search reveals 52 putative bacterial periplasmic heme-transporters, which can be grouped into 6 classes and most of which are associated with pathogenic bacteria. Multiple sequence alignment reveals that these putative heme-transporters have no conserved heme binding motif and share low sequence homology. A highly conserved tyrosine residue, Tyr 71, is likely to be an axial heme ligand in PhuT, which was confirmed by my site-directed mutagenesis and spectroscopic studies.
The first crystal structure of a periplasmic heme transport protein, PhuT, has been determined at 2.4-Å resolutions in collaboration with Poulos's laboratory (University of California, Irvine). The structure reveals two topologically similar globular domains and a long, rigid α-helix interdomain linker in PhuT. In agreement with my proposal, a ferric heme in PhuT is embedded in a narrow cleft between the N- and C-terminal domains and 5-coordinated with Tyr 71.
To investigate the important roles of Tyr 71 in heme binding and release and to explore pH-induced heme release mechanism, site-directed mutageneses of Tyr 71 to other common heme ligands (Met, Glu, Cys and His) were carried out. Heme-binding thermodynamics and heme-release kinetics studies indicate that O-based ligands (i.e. in the wild-type and the Y71E mutant PhuT) hold heme more tightly than the N- or S-based ligands (i.e. in the Y71H or Y71M variants), in agreement with the "HSAB" theory. Heme release under slightly acidic condition as revealed in the in vitro pH-dependent study suggests that protonations of axial heme ligand and other protein residues that involved in heme binding may contribute to the heme release process.
In order to characterize the other components in the heme uptake system, phuR, phuUV and phuW genes have been cloned into pET101D vector, respectively, and expressed in E. coli . The heme receptor PhuR has been purified as a 70 KDa protein. In vitro studies indicate that the wild-type PhuR not only binds free heme in the presence of 1% octyl-polyoxyethylene, but also assimilate heme from hemoglobin.
Förster Resonance Energy Transfer (FRET) technique has been applied to study heme binding/release and heme transport process. PhuT and PhuR were successfully labeled with Alexa Fluor® 488 via specifically engineered cysteine sites. The fluorescence intensity of the AF-donor is quenched when the protein binds to the acceptor molecule, heme or other metalloporphyrins (Pd-mP, SnP and ZnP), via a FRET process; the fluorescence signals can be recovered when heme (or the metalloporphyrins) is released from the proteins (by denaturation of the proteins, or transfer of heme to apomyoglobin). In vivo fluorescent labeling of PhuR in E. coliwith AF-488 has been achieved, which has a potential application to study the heme uptake process in live cells.
Tong, Yong, "Spectroscopic And Biochemical Studies On Novel Heme Transport Proteins From Pseudomonas Aeruginosa" (2010). Doctoral Dissertations 1896 - February 2014. 232.