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Function and cellular transport of iron chemistry

Chen, Chun-An
http://rave.ohiolink.edu/etdc/view?acc_num=osu1092775070

Abstract Details

2004, Doctor of Philosophy, Ohio State University, Chemistry.
In this research, we primarily focus on the structure and function of both the nucleotide binding domain, and the full length membrane-spanning transporter. First, the soluble nucleotide binding domain of Atm1 (Atm1-C), an ABC transporter in yeast mitochondria, that has previously been implicated in the maturation of cytosolic iron-sulfur cluster proteins, has been overexpressed in E. coli, purified, and characterized. The full length version of Atm1 from Saccharomyces cerevisiae has been cloned, over-expressed, purified from a yeast expression system, and characterized. A fluorescent assay of liposome-loaded reconstituted Atm1p suggested that Atm1p only allowed small molecules and/or metal complexes to cross the channel. Both pH gradient and fluorescent assays also indicated that ADP-bound Atm1p existed in an open state that is different from the closed state for ATP-bound Atm1p. The further discovery of an iron carrying peptide, hepcidin, provides the first step toward understanding iron trafficking in living cells. With eight, well-conserved cysteine residues in the sequence, hepcidin may not only be a signal peptide, but could potentially serve as an iron carrier. The iron binding properties have been determined by UV-vis spectroscopy, mass spectroscopy, and isothermal titration calorimetry (ITC). The iron binding affinity has been determined in the micromolar range. Studies by circular dichroism (CD) reveal varying degrees of secondary structure within an apparent dynamic tertiary fold. Taken together, hepcidin clearly binds iron, and the secondary structure change induced by iron binding may be required for the full function of the peptide in iron homeostasis and antimicrobial activity. In Part II, a novel fluorescent assay has been developed for monitoring the cleavage of a target RNA by cooper kanamycin in vivo. However, demonstration of the efficacy of such reagents in vitro is only a first step. To demonstrate in vivo cleavage chemistry we have designed a fluorescence assay based on use of the green fluorescent protein (GFP). The decrease in fluorescent intensity indicated the designed target RNA being destructed. The success in this assay provided a novel tool for screening a large quantity of drug molecules for their targets at the same time.
James Cowan (Advisor)
Chemistry, Biochemistry
Atm1p; ABC Transporter; Iron sulfur cluster; Hepcidin; HiPIP; Copper Kanamycin; R23

Recommended Citations

Citations

  • Chen, C.-A. (2004). Function and cellular transport of iron chemistry [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1092775070

    APA Style (7th edition)

  • Chen, Chun-An. Function and cellular transport of iron chemistry. 2004. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1092775070.

    MLA Style (8th edition)

  • Chen, Chun-An. "Function and cellular transport of iron chemistry." Doctoral dissertation, Ohio State University, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=osu1092775070

    Chicago Manual of Style (17th edition)

osu1092775070
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© 2004, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.