David N Silverman

University of Florida, Gainesville, Florida, United States

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Publications (182)759.3 Total impact

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    ABSTRACT: Biocatalytic CO 2 sequestration to reduce greenhouse-gas emissions from industrial processes is an active area of research. Carbonic anhydrases (CAs) are attractive enzymes for this process. However, the most active CAs display limited thermal and pH stability, making them less than ideal. As a result, there is an ongoing effort to engineer and/or find a thermostable CA to fulfill these needs. Here, the kinetic and thermal characterization is presented of an α-CA recently discovered in the mesophilic hydrothermal vent-isolate extremophile Thiomicrospira crunogena XCL-2 (TcruCA), which has a significantly higher thermostability compared with human CA II (melting temperature of 71.9°C versus 59.5°C, respectively) but with a tenfold decrease in the catalytic efficiency. The X-ray crystallographic structure of the dimeric TcruCA shows that it has a highly conserved yet compact structure compared with other α-CAs. In addition, TcruCA contains an intramolecular disulfide bond that stabilizes the enzyme. These features are thought to contribute significantly to the thermostability and pH stability of the enzyme and may be exploited to engineer α-CAs for applications in industrial CO 2 sequestration.
    No preview · Article · Aug 2015 · Acta Crystallographica Section D Biological Crystallography
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    ABSTRACT: Thiomicrospira crunogena XCL-2 expresses an α-carbonic anhydrase (TcruCA). Sequence alignments reveal that TcruCA displays a high sequence identity (>30%) relative to other α-CAs. This includes three conserved histidines that coordinate the active site zinc, a histidine proton shuttling residue, and opposing hydrophilic and hydrophobic sides that line the active site. The catalytic efficiency of TcruCA is considered moderate relative to other α-CAs (kcat/KM=1.1×10(7)M(-1)s(-1)), being a factor of ten less efficient than the most active α-CAs. TcruCA is also inhibited by anions with Cl(-), Br(-), and I(-), all showing Ki values in the millimolar range (53-361mM). Hydrogen sulfide (HS(-)) revealed the highest affinity for TcruCA with a Ki of 1.1μM. It is predicted that inhibition of TcruCA by HS(-) (an anion commonly found in the environment where Thiomicrospira crunogena is located) is a way for Thiomicrospira crunogena to regulate its carbon-concentrating mechanism (CCM) and thus the organism's metabolic functions. Results from this study provide preliminary insights into the role of TcruCA in the general metabolism of Thiomicrospira crunogena. Copyright © 2015. Published by Elsevier Ltd.
    No preview · Article · May 2015 · Bioorganic & medicinal chemistry letters
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    ABSTRACT: By using a combination of liquid and solid-state NMR spectroscopy, 15N-labeled 4-methylimidazole (4-MI) as a local probe of the environment has been studied: 1) in the polar, wet Freon CDF3/CDF2Cl down to 130 K, 2) in water at pH 12, and 3) in solid samples of the mutant H64A of human carbonic anhydrase II (HCA II). In the latter, the active-site His64 residue is replaced by alanine; the catalytic activity is, however, rescued by the presence of 4-MI. For the Freon solution, it is demonstrated that addition of water molecules not only catalyzes proton tautomerism but also lifts its quasidegeneracy. The possible hydrogen-bond clusters formed and the mechanism of the tautomerism are discussed. Information about the imidazole hydrogen-bond geometries is obtained by establishing a correlation between published 1H and 15N chemical shifts of the imidazole rings of histidines in proteins. This correlation is useful to distinguish histidines embedded in the interior of proteins and those at the surface, embedded in water. Moreover, evidence is obtained that the hydrogen-bond geometries of His64 in the active site of HCA II and of 4-MI in H64A HCA II are similar. Finally, the degeneracy of the rapid tautomerism of the neutral imidazole ring His64 reported by Shimahara et al. (J. Biol. Chem.­ 2007, 282, 9646) can be explained with a wet, polar, nonaqueous active-site conformation in the inward conformation, similar to the properties of 4-MI in the Freon solution. The biological implications for the enzyme mechanism are discussed.
    No preview · Article · Dec 2014 · Chemistry - A European Journal
  • Dayne West · Melissa A. Pinard · Chingkuang Tu · David N. Silverman · Robert McKenna
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    ABSTRACT: The binding of anions to carbonic anhydrase II (CA II) has been attributed to high affinity for the active-site zinc. An anion of interest is cyanate, for which contrasting binding modes have been reported in the literature. Previous spectroscopic data have shown cyanate behaving as an inhibitor, directly binding to the zinc, in contrast to previous crystallographic data that implied that cyanate acts as a substrate mimic that is not directly bound to the zinc but overlaps with the binding site of the substrate CO2. Wild-type and the V207I variant of CA II have been expressed and X-ray crystal structures of their cyanate complexes have been determined to 1.7 and 1.5 Å resolution, respectively. The rationale for the V207I CA II variant was its close proximity to the CO2-binding site. Both structures clearly show that the cyanate binds directly to the zinc. In addition, inhibition constants (∼40 µM) were measured using 18O-exchange mass spectrometry for wild-type and V207I CA II and were similar to those determined previously (Supuran et al., 1997). Hence, it is concluded that under the conditions of these experiments the binding of cyanate to CA II is directly to the zinc, displacing the zinc-bound solvent molecule, and not in a site that overlaps with the CO2 substrate-binding site.
    No preview · Article · Oct 2014 · Acta Crystallographica Section F: Structural Biology Communications
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    Mayank Aggarwal · Chingkuang Tu · David Silverman · Robert McKenna
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    ABSTRACT: Human carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the hydration and dehydration of CO2 and HCO3-, respectively. The reaction follows a ping-pong mechanism, where the rate limiting step is the transfer of a proton from the zinc-bound solvent out of the active site, via His64 which is widely believed to be the proton shuttling residue. Being involved in a number of physiological processes such as respiration, pH regulation, ureagenesis etc., CAs are therapeutic targets for inhibition to treat various diseases. However, the physiologically dominant isoform is CA II, which is catalytically highly efficient and is easily crystallizable. Thus, most of our knowledge in the design of CA inhibitors with pharmacological applications is based on detailed CA II crystallographic studies. The catalytic activity of a variant of CA II in which His64 is replaced with Ala (H64A CA II) can be enhanced by exogenous proton donors/acceptors, usually derivatives of imidazoles and pyridines. This article examines the mechanism through which this activity enhancement might occur. X-ray crystal structures of H64A CA II in complex with four imidazole derivatives have been determined and reveal multiple binding sites. We have identified two molecules of imidazoles that bind in region that is otherwise occupied by the "in" and "out" dual conformation of the side chain of His64 in wild-type CA II. The data presented here not only corroborates the importance of imidazole side chain of His64 in proton transfer during CA catalysis, but also provides a complete structural understanding of the mechanism by which imidazoles enhance (and inhibit when used in higher concentrations) the activity of H64A CA II. In addition to inhibition of CA by these imidazoles, the presence of a large number of binding sites also gives insights and preliminary data required to fragment addition approach of drug design against CA.
    Full-text · Article · Aug 2014 · Acta Crystallographica Section A: Foundations and Advances
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    ABSTRACT: Human carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the hydration and dehydration of CO2 and HCO3 −, respectively. The reaction follows a ping-pong mechanism, in which the rate-limiting step is the transfer of a proton from the zinc-bound solvent (OH−/H2O) in/out of the active site via His64, which is widely believed to be the proton-shuttling residue. The decreased catalytic activity (∼20-fold lower with respect to the wild type) of a variant of CA II in which His64 is replaced with Ala (H64A CA II) can be enhanced by exogenous proton donors/acceptors, usually derivatives of imidazoles and pyridines, to almost the wild-type level. X-ray crystal structures of H64A CA II in complex with four imidazole derivatives (imidazole, 1-­methylimidazole, 2-­methylimidazole and 4-methylimidazole) have been determined and reveal multiple binding sites. Two of these imidazole binding sites have been identified that mimic the positions of the ‘in’ and ‘out’ rotamers of His64 in wild-type CA II, while another directly inhibits catalysis by displacing the zinc-bound solvent. The data presented here not only corroborate the importance of the imidazole side chain of His64 in proton transfer during CA catalysis, but also provide a complete structural understanding of the mechanism by which imidazoles enhance (and inhibit when used at higher concentrations) the activity of H64A CA II.
    Full-text · Article · Mar 2014
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    ABSTRACT: The carbonic anhydrases (CAs; EC 4.2.1.1) are a family of metalloenzymes that catalyze the reversible hydration of carbon dioxide (CO2) and dehydration of bicarbonate (HCO3 (-)) in a two-step ping-pong mechanism: [Formula: see text] CAs are ubiquitous enzymes and are categorized into five distinct classes (α, β, γ, δ and ζ). The α-class is found primarily in vertebrates (and the only class of CA in mammals), β is observed in higher plants and some prokaryotes, γ is present only in archaebacteria whereas the δ and ζ classes have only been observed in diatoms.The focus of this chapter is on α-CAs as the structure-function relationship is best understood for this class, in particular for humans. The reader is referred to other reviews for an overview of the structure and catalytic mechanism of the other CA classes. The overall catalytic site structure and geometry of α-CAs are described in the first section of this chapter followed by the kinetic studies, binding of CO2, and the proton shuttle network.
    No preview · Article · Jan 2014 · Sub-cellular biochemistry
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    Full-text · Article · Oct 2013 · Neutron News
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    ABSTRACT: The presence of aromatic clusters has been found to be an integral feature of many proteins isolated from thermophilic microorganisms. Residues found in aromatic cluster interact via π-π or C-H···π bonds between the phenyl rings, which are among the weakest interactions involved in protein stability. The lone aromatic cluster in human carbonic anhydrase II (HCA II) is centered on F226 with the surrounding aromatics F66, F95 and W97 located 12 Å posterior the active site; a location which could facilitate proper protein folding and active site construction. The role of F226 in the structure, catalytic activity and thermostability of HCA II was investigated via site-directed mutagenesis of three variants (F226I/L/W) into this position. The measured catalytic rates of the F226 variants via (18)O-mass spectrometry were identical to the native enzyme, but differential scanning calorimetry studies revealed a 3-4 K decrease in their denaturing temperature. X-ray crystallographic analysis suggests that the structural basis of this destabilization is via disruption and/or removal of weak C-H···π interactions between F226 to F66, F95 and W97. This study emphasizes the importance of the delicate arrangement of these weak interactions among aromatic clusters in overall protein stability.
    No preview · Article · Sep 2013 · Archives of Biochemistry and Biophysics
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    ABSTRACT: The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration of CO2 to bicarbonate and a proton. Recently, there has been industrial interest in utilizing CAs as biocatalysts for carbon sequestration and biofuel production. The conditions used in these processes, however, result in high temperatures and acidic pH. This unfavorable environment results in rapid destabilization and loss of catalytic activity in CAs, ultimately resulting in cost-inefficient high-maintenance operation of the system. In order to negate these detrimental industrial conditions, cysteines at residues 23 (Ala23Cys) and 203 (Leu203Cys) were engineered into a wild-type variant of human CA II (HCAII) containing the mutation Cys206Ser. The X-ray crystallographic structure of the disulfide-containing HCAII (dsHCAII) was solved to 1.77 Å resolution and revealed that successful oxidation of the cysteine bond was achieved while also retaining desirable active-site geometry. Kinetic studies utilizing the measurement of (18)O-labeled CO2 by mass spectrometry revealed that dsHCAII retained high catalytic efficiency, and differential scanning calorimetry showed acid stability and thermal stability that was enhanced by up to 14 K compared with native HCAII. Together, these studies have shown that dsHCAII has properties that could be used in an industrial setting to help to lower costs and improve the overall reaction efficiency.
    No preview · Article · Aug 2013 · Acta Crystallographica Section D Biological Crystallography
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    ABSTRACT: Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277-298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20-25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site.
    No preview · Article · May 2013 · Acta Crystallographica Section D Biological Crystallography
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    ABSTRACT: Variants of human carbonic anhydrase II (HCA II) with amino-acid replacements at residues in contact with water molecules in the active-site cavity have provided insights into the proton transfer rates in this protein environment. X-ray crystallography and 18O exchange measured by membrane inlet mass spectrometry have been used to investigate structural and catalytic properties of variants of HCA II containing the replacements of Tyr7 with Phe (Y7F) and Asn67 with Gln (N67Q). The rate constants for proton transfer from His64 to the zinc-bound hydroxide in catalysis were 4 μs-1 and 9 μs-1 for Y7F and Y7F-N67Q, respectively, compared with a value of 0.8 μs-1 for wild-type HCA II. These higher values observed for Y7F and Y7F-N67Q HCA II could not be explained by differences in the values of the pKa of the proton donor (His64) and acceptor (zinc-bound hydroxide) or by orientation of the side chain of the proton shuttle residue His64. They appeared to be associated with reduced branching in the networks of hydrogen-bonded water molecules between the proton shuttle residue His64 and the zinc-bound solvent molecule as observed in crystal structures at 1.5 - 1.6 Å resolution. Moreover, Y7F-N67Q HCA II is unique among the variants studied in having a direct, hydrogen-bonded chain of water molecules between the zinc-bound solvent and Nδ of His64. This study provides the clearest example to date of the relevance of ordered water structure to rate constants for proton transfer in catalysis by carbonic anhydrase.
    No preview · Article · Dec 2012 · Biochemistry
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    ABSTRACT: Although widely distributed in Nature, only two class carbonic anhydrases are reported besides the founding member (Cam). Although roles for active-site residues important for catalysis have been identified in Cam, second shell residues have not been investigated. Two residues (Trp19 and Tyr200), positioned distant from the catalytic metal, were investigated by structural and kinetic analyses of replacement variants. Steady-state k(cat)/K(m) and k(cat) values decreased 3- to 10-fold for the Trp19 variants whereas the Y200 variants showed up to a 5-fold increase in k(cat). Rate constants for proton transfer decreased up to 10-fold for the Trp19 variants, and an increase of ∼2-fold for Y200F. The pK(a) values for the proton donor decreased 1-2 pH units for Trp19 and Y200 variants. The variant structures revealed a loop composed of residues 62-64 that occupies a different conformation than previously reported. The results show that, although Trp19 and Y200 are non-essential, they contribute to an extended active-site structure distant from the catalytic metal that fine tunes catalysis. Trp19 is important for both CO(2)/bicarbonate interconversion, and the proton transfer step of catalysis.
    No preview · Article · Oct 2012 · Archives of Biochemistry and Biophysics
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    ABSTRACT: This work examines the effect on catalysis of perturbing the position of bound CO2 in the active site of human carbonic anhydrase II (HCA II). Variants of HCA II replacing Val143 with hydrophobic residues, Ile, Leu, and Ala, were examined. The efficiency of catalysis in the hydration of CO2 for these variants was characterized by 18O exchange mass spectrometry, and their structures determined by X-ray crystallography at 1.7 to 1.5 Å resolution. The most hydrophobic substitutions V143I and V143L showed decreases in catalysis, as much as 20-fold, while the replacement by the smaller V143A showed only a moderate two-fold decrease in activity. Structural data for all three variants show no significant change in overall position of amino-acid side chains in the active site compared with wild type. However, V143A HCA II showed additional ordered water molecules in the active site compared to wild type. To further investigate the decrease in catalytic efficiency of V143I HCA II, an X-ray crystallographic CO2 entrapment experiment was performed to 0.93 Å resolution. This structure revealed an unexpected shift of the CO2 substrate towards the zinc bound solvent, placing it ~0.3 Ǻ closer than previously observed in wild type in conjunction with the observed dual occupancy of the product bicarbonate, presumably formed during the data acquisition. These data suggest that the Ile substitution at position 143 reduced catalytic efficiency is likely due to steric crowding resulting in destabilization of the transition state for conversion of CO2 into bicarbonate and a decreased product dissociation rate.
    No preview · Article · Oct 2012 · Biochemistry
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    ABSTRACT: Carbonic anhydrases (CAs) catalyze the hydration of CO(2) forming HCO(3)(-) and a proton, an important reaction for many physiological processes including respiration, fluid secretion, and pH regulation. As such, CA isoforms are prominent clinical targets for treating various diseases. The clinically used acetazolamide (AZM) is a sulfonamide that binds with high affinity to human CA isoform II (HCA II). There are several X-ray structures available of AZM bound to various CA isoforms, but these complexes do not show the charged state of AZM or the hydrogen atom positions of the protein and solvent. Neutron diffraction is a useful technique for directly observing H atoms and the mapping of H-bonding networks that can greatly contribute to rational drug design. To this end, the neutron structure of H/D exchanged HCA II crystals in complex with AZM was determined. The structure reveals the molecular details of AZM binding and the charged state of the bound drug. This represents the first determined neutron structure of a clinically used drug bound to its target.
    Full-text · Article · Aug 2012 · Journal of the American Chemical Society
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    ABSTRACT: Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. As such, there is enormous industrial interest in using CA as a bio-catalyst for carbon sequestration and biofuel production. However, to ensure cost-effective use of the enzyme under harsh industrial conditions, studies were initiated to produce variants with enhanced thermostability while retaining high solubility and catalytic activity. Kinetic and structural studies were conducted to determine the structural and functional effects of these mutations. X-ray crystallography revealed that a gain in surface hydrogen bonding contributes to stability while retaining proper active site geometry and electrostatics to sustain catalytic efficiency. The kinetic profiles determined under a variety of conditions show that the surface mutations did not negatively impact the carbon dioxide hydration or proton transfer activity of the enzyme. Together these results show that it is possible to enhance the thermal stability of human carbonic anhydrase II by specific replacements of surface hydrophobic residues of the enzyme. In addition, combining these stabilizing mutations with strategic active site changes have resulted in thermostable mutants with desirable kinetic properties.
    Full-text · Article · Jun 2012 · Protein Engineering Design and Selection
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    ABSTRACT: The carbonic anhydrases (CAs) in the α class are zinc-dependent metalloenzymes. Previous studies have reported that recombinant forms of carbonic anhydrase IX (CAIX), a membrane-bound form of CA expressed in solid tumors, appear to be activated by low levels of zinc independent of its well-studied role at the catalytic site. In this study, we sought to determine if CAIX is stimulated by zinc in its native environment. MDA-MB-231 breast cancer cells express CAIX in response to hypoxia. We compared CAIX activity associated with membrane ghosts isolated from hypoxic cells with that in intact hypoxic cells. We measured CA activity directly using (18)O exchange from (13)CO(2) into water determined by membrane inlet mass spectrometry. In membrane ghosts, there was little effect of zinc at low concentrations on CAIX activity, although at high concentration zinc was inhibitory. In intact cells, zinc had no significant effect on CAIX activity. This suggests that there is an appreciable decrease in sensitivity to zinc when CAIX is in its natural membrane milieu compared to the purified forms.
    Full-text · Article · Mar 2012 · Archives of Biochemistry and Biophysics
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    ABSTRACT: The tryptophan residue Trp5, highly conserved in the α class of carbonic anhydrases including human carbonic anhydrase II (HCA II), is positioned at the entrance of the active site cavity and forms a π-stacking interaction with the imidazole ring of the proton shuttle His64 in its outward orientation. We have observed that replacement of Trp5 in HCA II caused significant structural changes, as determined by X-ray diffraction, in the conformation of 11 residues at the N-terminus and in the orientation of the proton shuttle residue His64. Most significantly, two variants W5H and W5E HCA II had His64 predominantly outward in orientation, while W5F and wild type showed the superposition of both outward and inward orientations in crystal structures. Although Trp5 influences the orientation of the proton shuttle His64, this orientation had no significant effect on the rate constant for proton transfer near 1μs(-1), determined by exchange of (18)O between CO(2) and water measured by mass spectrometry. The apparent values of the pK(a) of the zinc-bound water and the proton shuttle residue suggest that different active-site conformations influence the two stages of catalysis, the proton transfer stage and the interconversion of CO(2) and bicarbonate.
    Full-text · Article · Dec 2011 · Archives of Biochemistry and Biophysics
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    ABSTRACT: The neutron structure of wild-type human carbonic anhydrase II at pH 7.8 has been determined to 2.0 angstrom resolution. Detailed analysis and comparison to the previously determined structure at pH 10.0 show important differences in the protonation of key catalytic residues in the active site as well as a rearrangement of the H-bonded water network For the first time, a completed H-bonded network stretching from the Zn-bound solvent to the proton shuttling residue, His64, has been directly observed.
    Full-text · Article · Nov 2011 · Biochemistry
  • Susan C. Frost · Ying Li · Chingkuang Tu · David N. Silverman

    No preview · Article · Jul 2011 · Cancer Research

Publication Stats

5k Citations
759.30 Total Impact Points

Institutions

  • 1976-2015
    • University of Florida
      • • Department of Pharmacology and Therapeutics
      • • Department of Biochemistry and Molecular Biology
      • • College of Medicine
      Gainesville, Florida, United States
  • 2010
    • Los Alamos National Laboratory
      • Bioscience Division
      Лос-Аламос, California, United States
  • 2007
    • Freie Universität Berlin
      • Institute of Chemistry and Biochemistry
      Berlin, Land Berlin, Germany
    • University of Utah
      • Department of Chemistry
      Salt Lake City, Utah, United States
  • 1993-2004
    • University of Toronto
      • • Department of Medical Biophysics
      • • Department of Chemistry
      Toronto, Ontario, Canada
  • 2002
    • Colgate University
      • Department of Chemistry
      Гамильтон, New York, United States
  • 1998-2001
    • University of Waterloo
      Ватерлоо, Ontario, Canada
    • University of Michigan
      Ann Arbor, Michigan, United States
  • 1995
    • University of Pennsylvania
      • Department of Chemistry
      Filadelfia, Pennsylvania, United States
    • Northwestern University
      • Department of Chemistry
      Evanston, Illinois, United States