Access Type
Open Access Dissertation
Date of Award
1-1-2009
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Chemistry
First Advisor
Mark R. Spaller
Second Advisor
Louis J. Romano
Abstract
Protein-mediated interactions that involve multiple ligands in their binding mechanisms are critical for many cellular functions. The primary focus of this dissertation research was to investigate such interactions for two proteins, the PDZ domain and frataxin, involving peptide and metal binding ligands, respectively. The three component projects of this work comprised (1) thermodynamic analysis of PDZ domain binding using calorimetry; (2) X-ray crystallographic structural studies of a PDZ dual domain; and (3) thermodynamic analysis of frataxin binding to iron. The specific goal of the research conducted with the PDZ domains was to understand the mechanism of action of multiple/tandem protein domains. The protein chosen for study was the dual domain PDZ1-2 of postsynaptic density-95 kDa (PSD-95) protein, a mammalian neuronal protein. The individual constructs, PDZ1 and PDZ2, were also prepared for comparative studies. Challenging the individual PDZ1 and PDZ2 and dual domains with short peptides derived form the C-terminal region of the domains' natural binding partners (8-mer to 6-mer) sequences, it was found that only one of the domains remains functional when expressed separately. Binding of PDZ1 and tandem PDZ1-2 could be characterized, whereas PDZ2 could not be stabilized under solution conditions to yield a valid binding curve. The use of bivalent peptides that would bind with 1:1 stoichiometry to the tandem PDZ1-2 domain was problematic due to ligand insolubility. In an ongoing effort to develop new cellular probes for in vivo investigation of multiprotein assembly, we studied modified ligand CN2180 which targets the tandem PDZ1-2 of PSD-95. Our in vitro testing by ITC of the same peptide after integrating the binding isoterm, yielded a stoichiometry of n=2, indicating that both binding sites are fully occupied, and dissociation constants for each of the two interaction sites were in the single digit micromolar range.
In terms of progress on that structural front, the dual PDZ domain protein construct PDZ1-2 of PSD-95 was successfully crystallized. Identifying the conditions required to grow crystals is a major step toward the goal of solving the structure of this tandem domain by X-ray methods. Optimization of the purification protocol resulted in high purity protein that allowed for increased volume of protein crystallization attempts. The optimized conditions yielded well-shaped hexagonal crystals for PDZ1-2, which crystallizes in cubic P23 group. Data reduction resulted in a Rmerge value of 11.2 %. A final structure could not be solved by molecular replacement methods, and additional work will be needed to accomplish this.
In the last project, calorimetry was employed to investigate the binding of frataxin to iron ions. This protein is essential for the effective regulation of cellular iron homeostasis. Frataxin, is essential for cellular iron control and is believed to serve as an iron chaperone that delivers mitochondrial Fe(II) to the enzymes ferrochelatase and the ISU apparatus for completion of heme and Fe-S cluster biosynthesis. Monomeric frataxin protein has a high affinity for ferrous ion, completely saturating at 2:1 iron to protein ratio. The micromolar dissociation constants measured for yeast frataxin, with respect to ferrous iron (Kd's of 2.0 and 3.0 uM) were obtained from calorimetric titrations. The weak interaction is consistent with the hypothesis of frataxin acting as an iron chaperone. Frataxin must be able to form a favorable interface with its protein partners and also easily release the metal. Although this thermodynamic binding study increases understanding of the underlying metal-binding behavior, additional structural characterization will be critical to help elucidate how frataxin binds iron and docks with its protein partners to promote metal delivery.
Recommended Citation
Jankovic, Ana, "Isothermal Titration Calorimetry Studies Of Protein-Mediated Interactions And Preliminary Structural Studies Of Tandem Pdz1-2 Domain Of Psd-95 Protein" (2009). Wayne State University Dissertations. 46.
https://digitalcommons.wayne.edu/oa_dissertations/46