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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
2R01ES012236-11
Application #
8625864
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Balshaw, David M
Project Start
2003-06-09
Project End
2019-02-28
Budget Start
2014-03-17
Budget End
2015-02-28
Support Year
11
Fiscal Year
2014
Total Cost
$293,524
Indirect Cost
$68,524
Name
University of Michigan Ann Arbor
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Plegaria, Jefferson S; Pecoraro, Vincent L (2016) De Novo Design of Metalloproteins and Metalloenzymes in a Three-Helix Bundle. Methods Mol Biol 1414:187-96
Plegaria, Jefferson S; Herrero, Christian; Quaranta, Annamaria et al. (2016) Electron transfer activity of a de novo designed copper center in a three-helix bundle fold. Biochim Biophys Acta 1857:522-30
Tebo, Alison G; Hemmingsen, Lars; Pecoraro, Vincent L (2015) Variable primary coordination environments of Cd(II) binding to three helix bundles provide a pathway for rapid metal exchange. Metallomics 7:1555-61
Ross, Matthew R; White, Aaron M; Yu, Fangting et al. (2015) Histidine Orientation Modulates the Structure and Dynamics of a de Novo Metalloenzyme Active Site. J Am Chem Soc 137:10164-76
Plegaria, Jefferson S; Dzul, Stephen P; Zuiderweg, Erik R P et al. (2015) Apoprotein Structure and Metal Binding Characterization of a de Novo Designed Peptide, α3DIV, that Sequesters Toxic Heavy Metals. Biochemistry 54:2858-73
Mocny, Catherine S; Pecoraro, Vincent L (2015) De novo protein design as a methodology for synthetic bioinorganic chemistry. Acc Chem Res 48:2388-96
Tebo, Alison G; Pecoraro, Vincent L (2015) Artificial metalloenzymes derived from three-helix bundles. Curr Opin Chem Biol 25:65-70
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

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