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 applicationis 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 (alpha-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 alpha-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)
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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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Ruckthong, Leela; Deb, Aniruddha; Hemmingsen, Lars et al. (2018) Incorporation of second coordination sphere D-amino acids alters Cd(II) geometries in designed thiolate-rich proteins. J Biol Inorg Chem 23:123-135
Tebo, Alison G; Pinter, Tyler B J; GarcĂ­a-Serres, Ricardo et al. (2018) Development of a Rubredoxin-Type Center Embedded in a de Dovo-Designed Three-Helix Bundle. Biochemistry 57:2308-2316
Koebke, Karl J; Pecoraro, Vincent L (2018) Development of de Novo Copper Nitrite Reductases: Where We Are and Where We Need To Go. ACS Catal 8:8046-8057
Koebke, Karl J; Yu, Fangting; Salerno, Elvin et al. (2018) Modifying the Steric Properties in the Second Coordination Sphere of Designed Peptides Leads to Enhancement of Nitrite Reductase Activity. Angew Chem Int Ed Engl 57:3954-3957
Cabral, Augusto C S; Jakovleska, Jovana; Deb, Aniruddha et al. (2018) Further insights into the metal ion binding abilities and the metalation pathway of a plant metallothionein from Musa acuminata. J Biol Inorg Chem 23:91-107
Koebke, Karl J; Ruckthong, Leela; Meagher, Jennifer L et al. (2018) Clarifying the Copper Coordination Environment in a de Novo Designed Red Copper Protein. Inorg Chem 57:12291-12302
Tebo, Alison G; Quaranta, Annamaria; Herrero, Christian et al. (2017) Intramolecular Photogeneration of a Tyrosine Radical in a Designed Protein. ChemPhotoChem 1:89-92
Ruckthong, Leela; Peacock, Anna F A; Pascoe, Cherilyn E et al. (2017) d-Cysteine Ligands Control Metal Geometries within De Novo Designed Three-Stranded Coiled Coils. Chemistry 23:8232-8243
Cangelosi, Virginia; Ruckthong, Leela; Pecoraro, Vincent L (2017) Lead(II) Binding in Natural and Artificial Proteins. Met Ions Life Sci 17:
Sgarlata, Carmelo; Giuffrida, Alessandro; Trivedi, Evan R et al. (2017) Anion Encapsulation Drives the Formation of Dimeric GdIII[15-metallacrown-5]3+ Complexes in Aqueous Solution. Inorg Chem 56:4771-4774

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