This CAREER award in the Inorganic, Bioinorganic and Organometallic Chemistry program supports research by Professor Sarah L. Michel at the University of Maryland Baltimore County (UMBC) to determine the consequences of iron substitution in zinc finger proteins. Iron has the potential to substitute for zinc in the metal binding sites of zinc finger proteins; however, this substitution may leave the zinc finger protein susceptible to oxidative damage. The proposed studies are aimed at unraveling the bioinorganic chemistry of iron substitution in zinc binding domains and the susceptibility of zinc finger proteins, with either zinc or iron coordinated, to oxidation. Two specific zinc finger proteins will be examined: tristetraprolin (TTP), an RNA-binding Cys3His-M(II) protein, and Neural Zinc Finger Factor 1 (NZF1), a DNA-binding Cys2HisCys-M(II) protein. The research aims to: 1) determine and quantify the factors that control the molecular recognition events between ferric and ferrous iron-substituted TTP and NZF1 and their RNA/DNA targets and 2) characterize the pathway of oxidative damage for iron substituted TTP and NZF1 and compare to the zinc bound and apo forms of the proteins.

An educational/research partnership between the pharmaceutical sciences department (PSC) at UMBC and the chemistry department at Morgan State University (MSU), a historically black college or university (HBCU) in Baltimore City will be developed. Undergraduates from MSU will participate in a week-long graduate school immersion program, SIMSI, that includes mentoring by PSC graduate students, a "Mini-Grad" school experience, skills building workshops and a career opportunities forum. MSU undergraduates will also participate in summer research experiences, online discussion groups with PSC mentors, and PSC department seminars. A DVD describing the partnership will be produced and made available to faculty at peer institutions. The aim of this partnership is to inspire undergraduate students from under-represented groups to pursue science careers by providing short and long term research experiences coupled with mentoring. PSC graduate students from the PI's laboratory will be involved in the partnership, and the group's work on iron substitution in zinc finger proteins will be the focus of one of the "Mini-Grad" school projects performed by MSU undergraduates. An ongoing research collaboration between Dr. Cymet at MSU and undergraduate students in her laboratory will enhance both the training of Dr. Cymet and her students but also the training of Dr. Michel's students. Undergraduate and graduate students working on the iron-zinc finger project will receive broad training in research at the interface of inorganic chemistry and biology.

Project Report

Zinc Finger (ZF) proteins are a large family of (mostly) eukaryotic proteins that regulate DNA and RNA. ZF’s utilize zinc as a structural element: zinc binds to a combination of four cysteine and/or histidine ligands in a near-tetrahedral coordination geometry resulting in a folded protein that can function (e.g. bind to DNA or RNA). Other divalent metal ions can also bind to ZFs, in vitro, including ferric and ferrous iron. Iron coordination to these ZFs has the potential to affect the function of these proteins as well as their susceptibility to oxidation; however, systematic studies in which the role(s) of iron coordination on ZF function are addressed are lacking. Thus, this project focused on (i) understanding the effects of iron substitution on ZF function and (ii) determining the susceptibility of ZF proteins, with either iron or zinc bound, to oxidation. These studies allowed us to obtain fundamental knowledge regarding how metal ion coordination affects ZF protein structure, function and reactivity. We initially focused on two ZFs, Tristetraprolin (or TTP), which has a Cys3His (or CCCH for 3 cysteine/one histidine) ligand set for metal ion coordination and Neural Zinc Finger Factor -1 (or NZF-1) which has a Cys2His2Cys (or CCHHC for 2 cysteines/ 2 histidines and one cysteine) ligand set. TTP regulates inflammation and NZF-1 regulates neuronal development and the normal physiology for both proteins exposes them to increased levels of iron and reactive oxygen species, making them excellent candidates to investigate the roles of iron and oxidation in function. We developed in vitro assays to measure metal ion coordination and RNA (for TTP) and DNA (for NZF-1) binding. We found that both proteins could coordinate iron and still retain selectivity for their oligonucleotide targets, suggesting that these proteins are flexible in their requirements for metal ions. The CCCH and CCHHC ZFs are new families of ZF proteins; and our new insights regarding iron coordination and function contribute to our general understanding of the functions of these new families of ZFs. We developed a rapid spectroscopic assay to measure oxidation of CCCH ZFs, which provided fundamental information regarding reactivity. We demonstrated that this oxidation assay is generalizable to any type of ZF, by studying oxidation of a CCHH type ZF. We expanded our studies to examine the role of cadmium coordination in the function of TTP, based upon compelling evidence that Cd coordination to TTP may be a mechanism for toxicity. We re-designed a ZF to have hydrolytic activity and developed the first generation of a hydrolytic ZF. Finally, we expanded our studies of CCCH and CCHHC ZFs to examine homologs with additional ZF domains to understand relationships between ZF domains, metal binding and DNA or RNA recognition. We developed ‘Spring into Maryland Science’ (SIMSI), a partnership with two Baltimore City colleges (Morgan State University, HBCU in Baltimore City and Notre Dame of Maryland University, Womens’ College in Baltimore City), to offer opportunities for talented undergraduates to participate in ‘mini-graduate school’ immersion experiences and provide mentoring by current graduate students. SIMSI participants have gone on to participate in summer research experiences and to PhD programs in chemistry and biology. The knowledge gained from our work has provided fundamental insight into how specific ZFs respond to metal ions and oxidants. Particularly important is the insight into relationships between metal ion coordination to ZF domains and DNA or RNA binding for newly discovered families of ZFs that has been obtained. In addition, a novel method to study the role of oxidation in ZF protein function that has the potential to be applied to many other families of ZF has been developed. The SIMSI educational partnership has provided students from groups under-represented in science the opportunity to participate in cutting-edge science and has provided current graduate students mentoring opportunities.

National Science Foundation (NSF)
Division of Chemistry (CHE)
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Timothy E. Patten
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University of Maryland Baltimore
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