Mei M Whittaker

Oregon Health and Science University, Portland, Oregon, United States

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Publications (50)174.02 Total impact

  • Mei M Whittaker, James W Whittaker
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    ABSTRACT: The Saccharomyces cerevisiae mitochondrial carrier YGR257Cp (Mtm1p) is an integral membrane protein that plays an essential role in mitochondrial iron homeostasis and respiratory functions, but its carrier substrate has not previously been identified. Large amounts of pure protein are required for biochemical characterization, including substrate screening. Functional complementation of a Saccharomyces knockout by expression of TwinStrep tagged YGR257Cp demonstrates that an affinity tag does not interfere with protein function, but the expression level in is very low. Heterologous expression in Pichia pastoris improves the yield but the product is heterogeneous. Expression has been screened in several E. coli hosts, optimizing yield by modifying induction conditions and supplementing with rare tRNAs to overcome codon bias in the eukaryotic gene. Detection of an additional N-terminal truncation product in E. coli reveals the presence of a secondary intracistronic translation initiation site, which can be eliminated by silent mutagenesis of an alternative (Leu) initiation codon, resulting in production of a single, full-length polypeptide (∼30% of the total protein) as insoluble inclusion bodies. Purified inclusion bodies were successfully refolded and affinity purified, yielding approximately 40 mg of pure, soluble product per liter of culture. Refolded YGR257Cp binds pyridoxal 5'-phosphate tightly (KD < 1 uM), supporting a new hypothesis that the mitochondrial carrier YGR237Cp and its homologs function as high affinity PLP transporters in mitochondria, providing the first evidence for this essential transport function in eukaryotes.
    Protein Expression and Purification 10/2013; · 1.43 Impact Factor
  • Mei M Whittaker, James W Whittaker
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    ABSTRACT: Human manganese superoxide dismutase (Sod2p) has been expressed in yeast and the protein purified from isolated yeast mitochondria, yielding both the metallated protein and the less stable apoprotein in a single chromatographic step. At 30 °C growth temperature, more than half of the purified enzyme is apoprotein that can be fully activated following reconstitution, while the remainder contains a mixture of manganese and iron. In contrast, only fully metallated enzyme was isolated from a similarly constructed yeast strain expressing the homologous yeast manganese superoxide dismutase. Both the manganese content and superoxide dismutase activity of the recombinant human enzyme increased with increasing growth temperatures. The dependence of in vivo metallation state on growth temperature resembles the in vitro thermal activation behavior of human manganese superoxide dismutase observed in previous studies. Partially metallated human superoxide dismutase is fully active in protecting yeast against superoxide stress produced by addition of paraquat to the growth medium. However, a splice variant of human manganese superoxide dismutase (isoform B) is expressed as insoluble protein in both Escherichia coli and yeast mitochondria and did not protect yeast against superoxide stress.
    Archives of Biochemistry and Biophysics 04/2012; 523(2):191-7. · 3.37 Impact Factor
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    ABSTRACT: Metal binding by apo-manganese superoxide dismutase (apo-MnSOD) is essential for functional maturation of the enzyme. Previous studies have demonstrated that metal binding by apo-MnSOD is conformationally gated, requiring protein reorganization for the metal to bind. We have now solved the X-ray crystal structure of apo-MnSOD at 1.9Å resolution. The organization of active site residues is independent of the presence of the metal cofactor, demonstrating that protein itself templates the unusual metal coordination geometry. Electrophoretic analysis of mixtures of apo- and (Mn₂)-MnSOD, dye-conjugated protein, or C-terminal Strep-tag II fusion protein reveals a dynamic subunit exchange process associated with cooperative metal binding by the two subunits of the dimeric protein. In contrast, (S126C) (SS) apo-MnSOD, which contains an inter-subunit covalent disulfide-crosslink, exhibits anti-cooperative metal binding. The protein concentration dependence of metal uptake kinetics implies that protein dissociation is involved in metal binding by the wild type apo-protein, although other processes may also contribute to gating metal uptake. Protein concentration dependent small-zone size exclusion chromatography is consistent with apo-MnSOD dimer dissociation at low protein concentration (K(D)=1×10⁻⁵ M). Studies on metal uptake by apo-MnSOD in Escherichia coli cells show that the protein exhibits similar behavior in vivo and in vitro.
    Archives of Biochemistry and Biophysics 10/2010; 505(2):213-25. · 3.37 Impact Factor
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    Mei M Whittaker, James W Whittaker
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    ABSTRACT: Metal uptake by the antioxidant defense metalloenzyme manganese superoxide dismutase (MnSOD) is an essential step in the functional maturation of the protein that is just beginning to be investigated in detail. We have extended earlier in vitro studies on metal binding by the dimeric Escherichia coli apo-MnSOD to investigate the mechanism of metal uptake by tetrameric human and Thermus thermophilus apo-MnSODs. Like the E. coli apo-MnSOD, these proteins also bind metal ions in vitro in a thermally activated, pH-sensitive process. However, metal uptake by the tetrameric apo-MnSODs exhibits a number of important differences. In particular, there is no indication of conformational gating requirement for metal binding for these proteins, and the reaction is first-order in metal ion. The high concentration of metal ion that is required to achieve physiologically relevant metallation rates for tetrameric human apo-MnSOD in vitro suggests the possibility that co-translational metal binding or chaperone interactions may be required in vivo.
    Archives of Biochemistry and Biophysics 09/2009; 491(1-2):69-74. · 3.37 Impact Factor
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    Mei M Whittaker, James W Whittaker
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    ABSTRACT: Metal uptake by apomanganese superoxide dismutase in vitro is a complex process exhibiting multiphase "gated" reaction kinetics and a striking sigmoidal temperature profile that has led to a model of conformationally gated metal binding, requiring conversion between "closed" and "open" forms. This work systematically explores the structural determinants of metal binding in both wild-type (WT) apoprotein and mutational variants as a test of mechanistic models. The pH dependence of metalation under physiological conditions (37 degrees C) shows it is linked to ionization of a single proton with a p K a of 7.7. Size exclusion chromatography demonstrates that the apoprotein is dimeric even when it is fully converted to the open form. The role of molecular motions in metal binding has been probed by using disulfide engineering to introduce covalent constraints into the protein. While restricting motion at domain interfaces has no effect, constraining the subunit interface significantly perturbs metal uptake but does not prevent the process. Mutagenesis of residues in the active site environment results in a dramatic shift in the transition temperature by as much as 20 degrees C or a loss of pH sensitivity. On the basis of these results, a mechanism for metal uptake by manganese superoxide dismutase involving reorientation of active site residues to form a metal entry channel is proposed.
    Biochemistry 11/2008; 47(44):11625-36. · 3.38 Impact Factor
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    Yuk-Ki Lee, Mei M Whittaker, James W Whittaker
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    ABSTRACT: Galactose oxidase is a metalloenzyme containing a novel metalloradical complex in its active site, comprised of a mononuclear copper ion associated with a protein free radical. The free radical has been shown to be localized on an intrinsic redox cofactor, 3'-(S-cysteinyl)tyrosine (Cys-Tyr), formed by a posttranslational covalent coupling of tyrosine and cysteine side chains in a self-processing reaction. The role of the thioether linkage in the function of the Cys-Tyr cofactor is unresolved, and some computational studies have suggested that the thioether substituent has a negligible effect on the properties of the tyrosyl free radical. In order to address this question experimentally, we have incorporated site-selectively labeled tyrosine ((2)H, (13)C, (17)O) into galactose oxidase using an engineered tyrosine auxotroph strain of Pichia pastoris . (33)S was also incorporated into the protein. EPR spectra for the Cys-Tyr(*) free radical in each of these isotopic variants were analyzed to extract nuclear hyperfine parameters for comparison with theoretical predictions, and the unpaired spin distribution in the free radical was reconstructed from the hyperfine data. These labeling studies allow the first comprehensive experimental evaluation of the effect of the thioether linkage on the properties of Cys-Tyr(*) and indicate that previous calculations significantly underestimated the contribution of this feature to the electronic ground state of the free radical.
    Biochemistry 06/2008; 47(25):6637-49. · 3.38 Impact Factor
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    ABSTRACT: High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an alpha-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4x10(4) U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions.
    Biochemical and Biophysical Research Communications 05/2007; 356(4):925-9. · 2.28 Impact Factor
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    ABSTRACT: Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide and hydrogen peroxide. In this study, unusual nonstoichiometric burst kinetics of the steady state reaction were observed and analyzed in detail, revealing that a reversible inactivation process occurs during turnover, associated with a slow isomerization of the substrate complex. We have investigated the underlying molecular mechanism of this kinetic behavior by preparing recombinant barley oxalate oxidase in three distinct oxidation states (Mn(II), Mn(III), and Mn(IV)) and producing a nonglycosylated variant for detailed biochemical and spectroscopic characterization. Surprisingly, the fully reduced Mn(II) form, which represents the majority of the as-isolated native enzyme, lacks oxalate oxidase activity, but the activity is restored by oxidation of the metal center to either Mn(III) or Mn(IV) forms. All three oxidation states appear to interconvert under turnover conditions, and the steady state activity of the enzyme is determined by a balance between activation and inactivation processes. In O(2)-saturated buffer, a turnover-based redox modification of the enzyme forms a novel superoxidized mononuclear Mn(IV) biological complex. An oxalate activation role for the catalytic metal ion is proposed based on these results.
    Journal of Biological Chemistry 04/2007; 282(10):7011-23. · 4.65 Impact Factor
  • Mei M Whittaker, James W Whittaker
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    ABSTRACT: The SCO2837 open-reading frame is located within the conserved central core region of the Streptomyces coelicolor A3(2) genome, which contains genes required for essential cellular functions. SCO2837 protein (SCO2837p) expressed by Pichia pastoris is a copper metalloenzyme, catalyzing the oxidation of simple alcohols to aldehydes and reduction of dioxygen to hydrogen peroxide. Distinct optical absorption spectra are observed for oxidized and one-electron reduced holoenzyme, and a free radical EPR signal is present in the oxidized apoprotein, characteristic of the Tyr-Cys redox cofactor previously reported for fungal secretory radical copper oxidases, galactose oxidase and glyoxal oxidase, with which it shares weak sequence similarity. SCO2837p was detected in the growth medium of both S. coelicolor and a recombinant expression host (Streptomyces lividans TK64) by Western blotting, with the expression level dependent on the nature of the carbon source. This represents the first characterized example of a prokaryotic radical copper oxidase.
    Archives of Biochemistry and Biophysics 09/2006; 452(2):108-18. · 3.37 Impact Factor
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    ABSTRACT: Oxalate oxidase (EC 1.2.3.4) catalyzes the conversion of oxalate and dioxygen to hydrogen peroxide and carbon dioxide. In this study, glycolate was used as a structural analogue of oxalate to investigate substrate binding in the crystalline enzyme. The observed monodentate binding of glycolate to the active site manganese ion of oxalate oxidase is consistent with a mechanism involving C-C bond cleavage driven by superoxide anion attack on a monodentate coordinated substrate. In this mechanism, the metal serves two functions: to organize the substrates (oxalate and dioxygen) and to transiently reduce dioxygen. The observed structure further implies important roles for specific active site residues (two asparagines and one glutamine) in correctly orientating the substrates and reaction intermediates for catalysis. Combined spectroscopic, biochemical, and structural analyses of mutants confirms the importance of the asparagine residues in organizing a functional active site complex.
    Journal of Biological Chemistry 04/2006; 281(10):6428-33. · 4.65 Impact Factor
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    ABSTRACT: The acquisition of a catalytic metal cofactor is an essential step in the maturation of every metalloenzyme, including manganese superoxide dismutase (MnSOD). In this study, we have taken advantage of the quenching of intrinsic protein fluorescence by bound metal ions to continuously monitor the metallation reaction of Escherichia coli MnSOD in vitro, permitting a detailed kinetic characterization of the uptake mechanism. Apo-MnSOD metallation kinetics are "gated", zero order in metal ion for both the native Mn2+ and a nonnative metal ion (Co2+) used as a spectroscopic probe to provide greater sensitivity to metal binding. Cobalt-binding time courses measured over a range of temperatures (35-50 degrees C) reveal two exponential kinetic processes (fast and slow phases) associated with metal binding. The amplitude of the fast phase increases rapidly as the temperature is raised, reflecting the fraction of Apo-MnSOD in an "open" conformation, and its temperature dependence allows thermodynamic parameters to be estimated for the "closed" to "open" conformational transition. The sensitivity of the metallated protein to exogenously added chelator decreases progressively with time, consistent with annealing of an initially formed metalloprotein complex (k anneal = 0.4 min(-1)). A domain-separation mechanism is proposed for metal uptake by apo-MnSOD.
    Biophysical Journal 02/2006; 90(2):598-607. · 3.67 Impact Factor
  • Mei M Whittaker, James W Whittaker
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    ABSTRACT: Strains of the methylotrophic yeast Pichia pastoris auxotrophic for the aromatic amino acids (tyrosine, phenylalanine, and tryptophan) have been constructed by targeted gene disruption for protein labeling applications. Three strains, with defects in ARO1 (coding for a homolog of the arom pentafunctional enzyme), ARO7 (coding for chorismate mutase), and TYR1 (coding for prephenate dehydrogenase), have been engineered in a P. pastoris ura3Delta1 parent strain using standard methods. The nutritional requirements of these auxotrophic strains have been characterized and their utility as expression hosts for labeling recombinant proteins has been demonstrated. All three strains show a surprising sensitivity to rich culture medium and must be grown in supplemented minimal medium. The tyr1::URA3 strain in particular is strongly inhibited by tryptophan, and to a lesser extent by phenylalanine, leucine, and isoleucine. Highly efficient incorporation of exogenously supplied amino acids by these three auxotroph strains has been demonstrated using recombinant galactose oxidase. Stereochemically pure l-amino acids and racemic d,l-mixtures serve nearly equally well to support protein expression and labeling. These strains allow efficient labeling of aromatic amino acids in recombinant proteins, supporting NMR structural biology and a wide range of other biophysical studies.
    Protein Expression and Purification 07/2005; 41(2):266-74. · 1.43 Impact Factor
  • Acta Crystallographica Section A - ACTA CRYSTALLOGR A. 01/2005; 61.
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    ABSTRACT: The fungal enzyme galactose oxidase is a radical copper oxidase that catalyzes the oxidation of a broad range of primary alcohols to aldehydes. Previous mechanistic studies have revealed a large substrate deuterium kinetic isotope effect on galactose oxidase turnover whose magnitude varies systematically over a series of substituted benzyl alcohols, reflecting a change in the character of the transition state for substrate oxidation. In this work, these detailed mechanistic studies have been extended using a series of stereospecifically monodeuterated substrates, including 1-O-methyl-alpha-D-galactose as well as unsubstituted benzyl alcohol and 3- and 4-methoxy and 4-nitrobenzyl derivatives. Synthesis of all of these substrates was based on oxidation of the alpha,alpha'-dideuterated alcohol to the corresponding (2)H-labeled aldehyde, followed by asymmetric hydroboration using alpha-pinene/9-BBN reagents to form the stereoisomeric alcohols. Products from enzymatic oxidation of each of these substrates were characterized by mass spectrometry to quantitatively evaluate the substrate dependence of the stereoselectivity of the catalytic reaction. For all of these substrates, the selectivity for pro-S hydrogen abstraction was at least 95%. This selectivity appears to be a direct consequence of constraints imposed by the enzyme on the orientation of substrates bearing a branched beta-carbon. Steady state analysis of kinetic isotope effects on V/K has resolved individual contributions from primary and alpha-secondary kinetic isotope effects in the reaction, providing a test for the involvement of an electron transfer redox equilibrium in the oxidation process. Multiple isotope effect measurements utilizing simultaneous labeling of the substrate and solvent have contributed to refinement of the relation between proton transfer and hydrogen atom transfer steps in substrate oxidation.
    Biochemistry 12/2004; 43(43):13683-93. · 3.38 Impact Factor
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    ABSTRACT: Escherichia coli apomanganese superoxide dismutase, prepared by removing the native metal ion under denaturing conditions, exhibits thermally triggered metal uptake behavior previously observed for thermophilic and hyperthermophilic superoxide dismutases but over a lower temperature range. Differential scanning calorimetry of aposuperoxide dismutase and metalated superoxide dismutase unfolding transitions has provided quantitative estimates of the metal binding affinities for manganese superoxide dismutase. The binding constant for Mn(II) (K(Mn(II)) = 3.2 x 10(8) m(-1)) is surprisingly low in light of the essentially irreversible metal binding characteristic of this family of proteins and indicates that metal binding and release processes are dominated by kinetic, rather than thermodynamic, constraints. The kinetic stability of the metalloprotein complex can be traced to stabilization by elements of the protein that are independent of the presence or absence of the metal ion reflected in the thermally triggered metalation characteristic of these proteins. Binding constants for Mn(III), Fe(II), and Fe(III) complexes were estimated using quasireversible values for the unfolding enthalpy and DeltaC(p) for apo-Mn superoxide dismutase and the observed T(m) values for unfolding the metalated species in the absence of denaturants. For manganese and iron complexes, an oxidation state-dependent binding affinity reflects the protein perturbation of the metal redox potential.
    Journal of Biological Chemistry 07/2004; 279(26):27339-44. · 4.65 Impact Factor
  • Mei M Whittaker, James W Whittaker
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    ABSTRACT: Galactose oxidase is a copper metalloenzyme containing a novel protein-derived redox cofactor in its active site, formed by cross-linking two residues, Cys228 and Tyr272. Previous studies have shown that formation of the tyrosyl-cysteine (Tyr-Cys) cofactor is a self-processing step requiring only copper and dioxygen. We have investigated the biogenesis of cofactor-containing galactose oxidase from pregalactose oxidase lacking the Tyr-Cys cross-link but having a fully processed N-terminal sequence, using both Cu(I) and Cu(II). Mature galactose oxidase forms rapidly following exposure of a pregalactose oxidase-Cu(I) complex to dioxygen (t(1/2) = 3.9s at pH7). In contrast, when Cu(II) is used in place of Cu(I) the maturation process requires several hours (t(1/2) = 5.1 h). EDTA prevents reaction of pregalactose oxidase with Cu(II) but does not interfere with the Cu(I)-dependent biogenesis reaction. The yield of cross-link corresponds to the amount of copper added, although a fraction of the pregalactose oxidase protein is unable to undergo this cross-linking reaction. The latter component, which may have an altered conformation, does not interfere with analysis of cofactor biogenesis at low copper loading. The biogenesis product has been quantitatively characterized, and mechanistic studies have been developed for the Cu(I)-dependent reaction, which forms oxidized, mature galactose oxidase and requires two molecules of O2. Transient kinetics studies of the biogenesis reaction have revealed a pH sensitivity that appears to reflect ionization of a protein group (pKa = 7.3) at intermediate pH resulting in a rate acceleration and protonation of an early oxygenated intermediate at lower pH competing with commitment to cofactor formation. These spectroscopic, kinetic, and biochemical results lead to new insights into the biogenesis mechanism.
    Journal of Biological Chemistry 07/2003; 278(24):22090-101. · 4.65 Impact Factor
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    ABSTRACT: X-ray crystallography of the nonheme manganese catalase from Lactobacillus plantarum (LPC) [Barynin, V.V., Whittaker, M.M., Antonyuk, S.V., Lamzin, V.S., Harrison, P.M., Artymiuk, P.J. & Whittaker, J.W. (2001) Structure9, 725-738] has revealed the structure of the dimanganese redox cluster together with its protein environment. The oxidized [Mn(III)Mn(III)] cluster is bridged by two solvent molecules (oxo and hydroxo, respectively) together with a micro 1,3 bridging glutamate carboxylate and is embedded in a web of hydrogen bonds involving an outer sphere tyrosine residue (Tyr42). A novel homologous expression system has been developed for production of active recombinant LPC and Tyr42 has been replaced by phenylalanine using site-directed mutagenesis. Spectroscopic and structural studies indicate that disruption of the hydrogen-bonded web significantly perturbs the active site in Y42F LPC, breaking one of the solvent bridges and generating an 'open' form of the dimanganese cluster. Two of the metal ligands adopt alternate conformations in the crystal structure, both conformers having a broken solvent bridge in the dimanganese core. The oxidized Y42F LPC exhibits strong optical absorption characteristic of high spin Mn(III) in low symmetry and lower coordination number. MCD and EPR measurements provide complementary information defining a ferromagnetically coupled electronic ground state for a cluster containing a single solvent bridge, in contrast to the diamagnetic ground state found for the native cluster containing a pair of solvent bridges. Y42F LPC has less than 5% of the catalase activity and much higher Km for H2O2 ( approximately 1.4 m) at neutral pH than WT LPC, although the activity is slightly restored at high pH where the cluster is converted to a diamagnetic form. These studies provide new insight into the contribution of the outer sphere tyrosine to the stability of the dimanganese cluster and the role of the solvent bridges in catalysis by dimanganese catalases.
    European Journal of Biochemistry 03/2003; 270(6):1102-16. · 3.58 Impact Factor
  • Journal of The American Chemical Society - J AM CHEM SOC. 04/2002; 114(10).
  • James W. Whittaker, Mei M. Whittaker
    Journal of the American Chemical Society 04/2002; 113(15). · 10.68 Impact Factor
  • Mei M. Whittaker, Yao Yuan Chuang, James W. Whittaker
    Journal of The American Chemical Society - J AM CHEM SOC. 04/2002; 115(22).

Publication Stats

923 Citations
174.02 Total Impact Points

Institutions

  • 2001–2013
    • Oregon Health and Science University
      • • Department of Environmental and Biomolecular Systems
      • • Institute of Environmental Health
      • • Department of Biochemistry & Molecular Biology
      Portland, Oregon, United States
    • The University of Sheffield
      • Department of Molecular Biology and Biotechnology
      Sheffield, ENG, United Kingdom
  • 2004
    • Shriners Hospitals for Children
      Tampa, Florida, United States
  • 1997–2000
    • Oregon Institute of Technology
      Wilsonville, Oregon, United States
  • 1988–2000
    • Carnegie Mellon University
      • Department of Chemistry
      Pittsburgh, Pennsylvania, United States
  • 1994
    • University of Michigan
      • Department of Chemistry
      Ann Arbor, MI, United States