Despite widespread detrimental human health effects due to heavy metal toxicity and neurodegenerative morbidity associated with dysfunctional metal homeostasis, a detailed molecular understanding of heavy metal protein interactions has yet to be attained. Fundamental chemical questions regarding toxic metal properties remain because these systems are often too complex, transient, or insoluble to be probed in a way that yields useful information. Our long-term goal is to resolve the processes by which toxic metals interact with proteins leading to morbidity or mortality. The overall objective of this application is to use an innovative approach, de novo protein design, to assess thermodynamics and kinetics for toxic metals binding to proteins and provide new spectroscopic correlations to enhance characterization of toxic metal-protein interactions significantly. Our central hypothesis is that our well-defined de novo designed metalloproteins (?-helical 3-stranded coiled coils and 3-helix bundles) will provide detailed insight into toxic metal chemistry that can be applied to understand more complex systems. Our basic premise is that de novo protein design provides simple, highly-controllable scaffolds well-suited to extract fundamental information on toxic metal chemistry and yield key insight into molecular function by systematically examining different coordination sites in aqueous peptidic environments. The rationale of the proposed research is that it will provide new information on protein-toxic metal interactions that have eluded the scrutiny of other approaches. Guided by strong preliminary data, our hypothesis will be tested through three Specific Aims: 1) Prepare asymmetric metal binding sites in designed proteins;2) Use designed proteins to develop spectroscopic characterization of metal-protein interactions;and 3) Use new protein designs to characterize toxic metal dynamics and thermodynamics in proteins.
Aim 1 applies three approaches (Pb-assisted assembly, covalent linkage of peptides, and inherent asymmetry within an ?-helical bundle) to obtaining asymmetric metal binding sites. This will allow study of heteroleptic toxic metal sites in Aims 2 and 3.
Aim 2 further develops spectroscopic methods (113Cd NMR, 111mCd PAC, 207Pb NMR, 204mPb PAC) with our well-defined metal sites, then applies our correlations to confidently assign metal sites in more complex natural systems.
Aim 3 characterizes toxic metal thermodynamics and kinetics by analyzing how Pb(II), As(III), and Cd(II) are inserted into our designed peptides and compares their binding constants with those for Fe(III), Cu(I/II), and Zn(II). Our research will provide vital structural characterization, binding constants, and kinetic studies while developing methods for probing protein-bound toxic metals. Our research is significant because clarification of thermodynamic and kinetic metal recognition processes will yield predictive power over how proteins are targeted and innovative because we are using a non-traditional approach, de novo protein design, to answer questions that cannot be fully addressed by direct studies on native biochemical systems or by synthesizing small molecule model complexes.

Public Health Relevance

Heavy metals such as Pb(II), Hg(II), As(III) and Cd(II) cause severe health concerns due both to their acute toxicity and the long term effects of chronic exposure (e.g., the EPA estimated in 2002 that over 300,000 children in the USA had blood levels exceeding 10 ug/dL). Our studies address these concerns in numerous ways: including making mimics of heavy metal binding sites in proteins in order to understand how heavy metals bind to proteins, defining the rates at which reactions occur and establishing the thermodynamic preferences of these metals to different sites. Studies such as these clarify the molecular mechanism for heavy metal molecular recognition by proteins.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-B (02))
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Balshaw, David M
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University of Michigan Ann Arbor
Schools of Arts and Sciences
Ann Arbor
United States
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Cangelosi, Virginia M; Deb, Aniruddha; Penner-Hahn, James E et al. (2014) A de?novo designed metalloenzyme for the hydration of CO2. Angew Chem Int Ed Engl 53:7900-3
Zastrow, Melissa L; Pecoraro, Vincent L (2014) Designing hydrolytic zinc metalloenzymes. Biochemistry 53:957-78
Peacock, Anna F A; Pecoraro, Vincent L (2013) Natural and artificial proteins containing cadmium. Met Ions Life Sci 11:303-37
Luczkowski, Marek; Zeider, Brian A; Hinz, Alia V H et al. (2013) Probing the coordination environment of the human copper chaperone HAH1: characterization of Hg(II)-bridged homodimeric species in solution. Chemistry 19:9042-9
Zastrow, Melissa L; Pecoraro, Vincent L (2013) Designing functional metalloproteins: from structural to catalytic metal sites. Coord Chem Rev 257:2565-2588
Zastrow, Melissa L; Pecoraro, Vincent L (2013) Influence of active site location on catalytic activity in de novo-designed zinc metalloenzymes. J Am Chem Soc 135:5895-903
Chakraborty, Saumen; Iranzo, Olga; Zuiderweg, Erik R P et al. (2012) Experimental and theoretical evaluation of multisite cadmium(II) exchange in designed three-stranded coiled-coil peptides. J Am Chem Soc 134:6191-203
Zastrow, Melissa L; Peacock, Anna F A; Stuckey, Jeanne A et al. (2012) Hydrolytic catalysis and structural stabilization in a designed metalloprotein. Nat Chem 4:118-23
Zampella, Giuseppe; Neupane, Kosh P; De Gioia, Luca et al. (2012) The importance of stereochemically active lone pairs for influencing Pb(II) and As(III) protein binding. Chemistry 18:2040-50
Neupane, Kosh P; Pecoraro, Vincent L (2011) Pb-207 NMR spectroscopy reveals that Pb(II) coordinates with glutathione (GSH) and tris cysteine zinc finger proteins in a PbS3 coordination environment. J Inorg Biochem 105:1030-4

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