Francis E Jenney

Philadelphia College of Osteopathic Medicine, Philadelphia, Pennsylvania, United States

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Publications (68)417.88 Total impact

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    ABSTRACT: The active site of [NiFe] hydrogenase is an excellent catalyst for hydrogen conversion with an intriguing ability to recover from exposure to dioxygen. In their Communication on page 724 ff., W. Lubitz, S. P. Cramer, and co‐workers use the synchrotron technique of nuclear resonance vibrational spectroscopy to identify normal modes associated with active‐site FeCN and FeCO motion.
    Angewandte Chemie International Edition 01/2013; 52(2). DOI:10.1002/anie.201208498 · 11.34 Impact Factor
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    ABSTRACT: Nuclear inelastic scattering of (57) Fe labeled [NiFe] hydrogenase is shown to give information on different states of the enzyme. It was thus possible to detect and assign Fe-CO and Fe-CN bending and stretching vibrations of the active site outside the spectral range of the Fe-S cluster normal modes.
    Angewandte Chemie International Edition 01/2013; DOI:10.1002/anie.201204616 · 11.34 Impact Factor
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    Angewandte Chemie 01/2013; 125(2). DOI:10.1002/ange.201208498
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    ABSTRACT: C-1 carriers are essential cofactors in all domains of life, and in Archaea, these can be derivatives of tetrahydromethanopterin (H(4)-MPT) or tetrahydrofolate (H(4)-folate). Their synthesis requires 6-hydroxymethyl-7,8-dihydropterin diphosphate (6-HMDP) as the precursor, but the nature of pathways that lead to its formation were unknown until the recent discovery of the GTP cyclohydrolase IB/MptA family that catalyzes the first step, the conversion of GTP to dihydroneopterin 2',3'-cyclic phosphate or 7,8-dihydroneopterin triphosphate [El Yacoubi, B.; et al. (2006) J. Biol. Chem., 281, 37586-37593 and Grochowski, L. L.; et al. (2007) Biochemistry46, 6658-6667]. Using a combination of comparative genomics analyses, heterologous complementation tests, and in vitro assays, we show that the archaeal protein families COG2098 and COG1634 specify two of the missing 6-HMDP synthesis enzymes. Members of the COG2098 family catalyze the formation of 6-hydroxymethyl-7,8-dihydropterin from 7,8-dihydroneopterin, while members of the COG1634 family catalyze the formation of 6-HMDP from 6-hydroxymethyl-7,8-dihydropterin. The discovery of these missing genes solves a long-standing mystery and provides novel examples of convergent evolutions where proteins of dissimilar architectures perform the same biochemical function.
    ACS Chemical Biology 08/2012; 7(11). DOI:10.1021/cb300342u · 5.36 Impact Factor
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    ABSTRACT: The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H(2) production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production.
    Journal of Biological Chemistry 12/2011; 287(5):3257-64. DOI:10.1074/jbc.M111.290916 · 4.60 Impact Factor
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    ABSTRACT: Hydrogen gas is an attractive alternative fuel as it is carbon neutral and has higher energy content per unit mass than fossil fuels. The biological enzyme responsible for utilizing molecular hydrogen is hydrogenase, a heteromeric metalloenzyme requiring a complex maturation process to assemble its O(2)-sensitive dinuclear-catalytic site containing nickel and iron atoms. To facilitate their utility in applied processes, it is essential that tools are available to engineer hydrogenases to tailor catalytic activity and electron carrier specificity, and decrease oxygen sensitivity using standard molecular biology techniques. As a model system we are using hydrogen-producing Pyrococcus furiosus, which grows optimally at 100°C. We have taken advantage of a recently developed genetic system that allows markerless chromosomal integrations via homologous recombination. We have combined a new gene marker system with a highly-expressed constitutive promoter to enable high-level homologous expression of an engineered form of the cytoplasmic NADP-dependent hydrogenase (SHI) of P. furiosus. In a step towards obtaining 'minimal' hydrogenases, we have successfully produced the heterodimeric form of SHI that contains only two of the four subunits found in the native heterotetrameric enzyme. The heterodimeric form is highly active (150 units mg(-1) in H(2) production using the artificial electron donor methyl viologen) and thermostable (t(1/2) ∼0.5 hour at 90°C). Moreover, the heterodimer does not use NADPH and instead can directly utilize reductant supplied by pyruvate ferredoxin oxidoreductase from P. furiosus. The SHI heterodimer and POR therefore represent a two-enzyme system that oxidizes pyruvate and produces H(2) in vitro without the need for an intermediate electron carrier.
    PLoS ONE 10/2011; 6(10):e26569. DOI:10.1371/journal.pone.0026569 · 3.53 Impact Factor
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    ABSTRACT: We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study oxidized and reduced forms of the [4Fe-4S] cluster in the D14C variant ferredoxin from Pyrococcus furiosus (Pf D14C Fd). To assist the normal-mode assignments, we conducted NRVS with D14C ferredoxin samples with (36)S substituted into the [4Fe-4S] cluster bridging sulfide positions, and a model compound without ligand side chains, (Ph(4)P)(2)[Fe(4)S(4)Cl(4)]. Several distinct regions of NRVS intensity are identified, ranging from "protein" and torsional modes below 100 cm(-1), through bending and breathing modes near 150 cm(-1), to strong bands from Fe-S stretching modes between 250 and ∼400 cm(-1). The oxidized ferredoxin samples were also investigated by resonance Raman (RR) spectroscopy. We found good agreement between NRVS and RR frequencies, but because of different selection rules, the intensities vary dramatically between the two types of spectra. The (57)Fe partial vibrational densities of states for the oxidized samples were interpreted by normal-mode analysis with optimization of Urey-Bradley force fields for local models of the [4Fe-4S] clusters. Full protein model calculations were also conducted using a supplemented CHARMM force field, and these calculations revealed low-frequency modes that may be relevant to electron transfer with Pf Fd partners. Density functional theory (DFT) calculations complemented these empirical analyses, and DFT was used to estimate the reorganization energy associated with the [Fe(4)S(4)](2+/+) redox cycle. Overall, the NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.
    Biochemistry 06/2011; 50(23):5220-35. DOI:10.1021/bi200046p · 3.19 Impact Factor
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    ABSTRACT: In attempts to develop a method of introducing DNA into Pyrococcus furiosus, we discovered a variant within the wild-type population that is naturally and efficiently competent for DNA uptake. A pyrF gene deletion mutant was constructed in the genome, and the combined transformation and recombination frequencies of this strain allowed marker replacement by direct selection using linear DNA. We have demonstrated the use of this strain, designated COM1, for genetic manipulation. Using genetic selections and counterselections based on uracil biosynthesis, we generated single- and double-deletion mutants of the two gene clusters that encode the two cytoplasmic hydrogenases. The COM1 strain will provide the basis for the development of more sophisticated genetic tools allowing the study and metabolic engineering of this important hyperthermophile.
    Applied and Environmental Microbiology 02/2011; 77(7):2232-8. DOI:10.1128/AEM.02624-10 · 3.95 Impact Factor
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    ABSTRACT: Metal-containing proteins comprise a diverse and sizable category within the proteomes of organisms, ranging from proteins that use metals to catalyze reactions to proteins in which metals play key structural roles. Unfortunately, reliably predicting that a protein will contain a specific metal from its amino acid sequence is not currently possible. We recently developed a generally-applicable experimental technique for finding metalloproteins on a genome-wide scale. Applying this metal-directed protein purification approach (ICP-MS and MS/MS based) to the prototypical microbe Pyrococcus furiosus conclusively demonstrated the extent and diversity of the uncharacterized portion of microbial metalloproteomes since a majority of the observed metal peaks could not be assigned to known or predicted metalloproteins. However, even using this technique, it is not technically feasible to purify to homogeneity all metalloproteins in an organism. In order to address these limitations and complement the metal-directed protein purification, we developed a computational infrastructure and statistical methodology to aid in the pursuit and identification of novel metalloproteins. We demonstrate that our methodology enables predictions of metal-protein interactions using an experimental data set derived from a chromatography fractionation experiment in which 870 proteins and 10 metals were measured over 2,589 fractions. For each of the 10 metals, cobalt, iron, manganese, molybdenum, nickel, lead, tungsten, uranium, vanadium, and zinc, clusters of proteins frequently occurring in metal peaks (of a specific metal) within the fractionation space were defined. This resulted in predictions that there are from 5 undiscovered vanadium- to 13 undiscovered cobalt-containing proteins in Pyrococcus furiosus. Molybdenum and nickel were chosen for additional assessment producing lists of genes predicted to encode metalloproteins or metalloprotein subunits, 22 for nickel including seven from known nickel-proteins, and 20 for molybdenum including two from known molybdo-proteins. The uncharacterized proteins are prime candidates for metal-based purification or recombinant approaches to validate these predictions. We conclude that the largely uncharacterized extent of native metalloproteomes can be revealed through analysis of the co-occurrence of metals and proteins across a fractionation space. This can significantly impact our understanding of metallobiochemistry, disease mechanisms, and metal toxicity, with implications for bioremediation, medicine and other fields.
    BMC Bioinformatics 02/2011; 12:64. DOI:10.1186/1471-2105-12-64 · 2.67 Impact Factor
  • Francis E. Jenney, Michael W. W. Adams
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    ABSTRACT: Metalloproteins play critical roles in living cells. There are more than a dozen metals with known or suspected biological functions, which include determining structure, electron transfer, and catalysis. The binding of a metal cofactor can generate beautifully colored proteins, but, more importantly, these metalloproteins function in essentially every metabolic pathway, greatly extending the chemistry past those that are available to a living cell with only the side groups of the 20 amino acids. The number and types of metalloproteins in cells remain surprisingly underappreciated, and only recently, with the advent of coupled purification and detection techniques, such as capillary electrophoresis and inductively coupled plasma emission mass spectrometry, is the true extent of the metalloproteome being revealed. Proteins from hyperthermophilic microorganisms are of particular utility for studying metalloproteins due to their extreme stability and relative ease of purification, both from native and recombinant sources. Herein we focus on the metalloproteins that have been characterized from some model hyperthermophilic systems with an emphasis on their properties and the roles that they play in primary metabolism.
    Extremophiles Handbook, 01/2011: pages 521-545; , ISBN: 978-4-431-53897-4
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    ABSTRACT: Metal ion cofactors afford proteins virtually unlimited catalytic potential, enable electron transfer reactions and have a great impact on protein stability. Consequently, metalloproteins have key roles in most biological processes, including respiration (iron and copper), photosynthesis (manganese) and drug metabolism (iron). Yet, predicting from genome sequence the numbers and types of metal an organism assimilates from its environment or uses in its metalloproteome is currently impossible because metal coordination sites are diverse and poorly recognized. We present here a robust, metal-based approach to determine all metals an organism assimilates and identify its metalloproteins on a genome-wide scale. This shifts the focus from classical protein-based purification to metal-based identification and purification by liquid chromatography, high-throughput tandem mass spectrometry (HT-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) to characterize cytoplasmic metalloproteins from an exemplary microorganism (Pyrococcus furiosus). Of 343 metal peaks in chromatography fractions, 158 did not match any predicted metalloprotein. Unassigned peaks included metals known to be used (cobalt, iron, nickel, tungsten and zinc; 83 peaks) plus metals the organism was not thought to assimilate (lead, manganese, molybdenum, uranium and vanadium; 75 peaks). Purification of eight of 158 unexpected metal peaks yielded four novel nickel- and molybdenum-containing proteins, whereas four purified proteins contained sub-stoichiometric amounts of misincorporated lead and uranium. Analyses of two additional microorganisms (Escherichia coli and Sulfolobus solfataricus) revealed species-specific assimilation of yet more unexpected metals. Metalloproteomes are therefore much more extensive and diverse than previously recognized, and promise to provide key insights for cell biology, microbial growth and toxicity mechanisms.
    Nature 08/2010; 466(7307):779-82. DOI:10.1038/nature09265 · 42.35 Impact Factor
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    ABSTRACT: We have applied (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to identify protein-bound dinitrosyl iron complexes. Intense NRVS peaks due to vibrations of the N-Fe-N unit can be observed between 500 and 700 cm(-1) and are diagnostic indicators of the type of iron dinitrosyl species present. NRVS spectra for four iron dinitrosyl model compounds are presented and used as benchmarks for the identification of species formed in the reaction of Pyrococcus furiosus ferredoxin D14C with nitric oxide.
    Journal of the American Chemical Society 05/2010; 132(20):6914-6. DOI:10.1021/ja101002f · 11.44 Impact Factor
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    ABSTRACT: Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins). Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2). The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins.
    PLoS ONE 05/2010; 5(5):e10526. DOI:10.1371/journal.pone.0010526 · 3.53 Impact Factor
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    ABSTRACT: Pyrococcus furiosus is a shallow marine, anaerobic archaeon that grows optimally at 100 degrees C. Addition of H(2)O(2) (0.5 mM) to a growing culture resulted in the cessation of growth with a 2-h lag before normal growth resumed. Whole genome transcriptional profiling revealed that the main response occurs within 30 min of peroxide addition, with the up-regulation of 62 open reading frames (ORFs), 36 of which are part of 10 potential operons. More than half of the up-regulated ORFs are of unknown function, while some others encode proteins that are involved potentially in sequestering iron and sulfide, in DNA repair and in generating NADPH. This response is thought to involve primarily damage repair rather than protection, since cultures exposed to sub-toxic levels of H(2)O(2) were not more resistant to the subsequent addition of H(2)O(2) (0.5-5.0 mM). Consequently, there is little if any induced protective response to peroxide. The organism maintains a constitutive protective mechanism involving high levels of oxidoreductase-type enzymes such as superoxide reductase, rubrerythrin, and alkyl hydroperoxide reductase. Related hyperthermophiles contain homologs of the proteins involved in the constitutive protective mechanism but these organisms were more sensitive to peroxide than P. furiosus and lack several of its peroxide-responsive ORFs.
    Archives of Microbiology 04/2010; 192(6):447-59. DOI:10.1007/s00203-010-0570-z · 1.86 Impact Factor
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    ABSTRACT: We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.
    Nature Methods 09/2009; 6(8):606-12. DOI:10.1038/nmeth.1353 · 25.95 Impact Factor
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    ABSTRACT: Virtually all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes, the compositions of which are largely undefined. They cannot be predicted solely from bioinformatics analyses nor are there well defined techniques currently available to unequivocally identify protein complexes (PCs). To address this issue, we attempted to directly determine the identity of PCs from native microbial biomass using Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100 degrees C, as the model organism. Novel PCs were identified by large scale fractionation of the native proteome using non-denaturing, sequential column chromatography under anaerobic, reducing conditions. A total of 967 distinct P. furiosus proteins were identified by mass spectrometry (nano LC-ESI-MS/MS), representing approximately 80% of the cytoplasmic proteins. Based on the co-fractionation of proteins that are encoded by adjacent genes on the chromosome, 106 potential heteromeric PCs containing 243 proteins were identified, only 20 of which were known or expected. In addition to those of unknown function, novel and uncharacterized PCs were identified that are proposed to be involved in the metabolism of amino acids (10), carbohydrates (four), lipids (two), vitamins and metals (three), and DNA and RNA (nine). A further 30 potential PCs were classified as tentative, and the remaining potential PCs (13) were classified as weakly interacting. Some major advantages of native biomass fractionation for PC identification are that it provides a road map for the (partial) purification of native forms of novel and uncharacterized PCs, and the results can be utilized for the recombinant production of low abundance PCs to provide enough material for detailed structural and biochemical analyses.
    Molecular &amp Cellular Proteomics 12/2008; 8(4):735-51. DOI:10.1074/mcp.M800246-MCP200 · 7.25 Impact Factor
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    ABSTRACT: Hydrothermal microbiotopes are characterized by the consumption and production of molecular hydrogen. Heterotrophic hyperthermophilic microorganisms (growth T(opt)> or =80 degrees C) actively participate in the production of H(2) in these environments through the fermentation of peptides and carbohydrates. Hyperthermophiles have been shown to approach the theoretical (Thauer) limit of 4 mol of H(2) produced per mole of glucose equivalent consumed, albeit at lower volumetric productivities than observed for mesophilic bacteria, especially enterics and clostridia. Potential advantages for biohydrogen production at elevated temperatures include fewer metabolic byproducts formed, absence of catabolic repression for growth on heterogeneous biomass substrates, and reduced loss of H(2) through conversion to H(2)S and CH(4) by mesophilic consortia containing sulfate reducers and methanogens. To fully exploit the use of these novel microorganisms and their constituent hydrogenases for biohydrogen production, development of versatile genetic systems and improvements in current understanding of electron flux from fermentable substrates to H(2) in hyperthermophiles are needed.
    Metabolic Engineering 11/2008; 10(6):394-404. DOI:10.1016/j.ymben.2008.06.007 · 8.26 Impact Factor
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    ABSTRACT: Key enzymes involved in end-product formation were identified in Thermoanaerobacterium saccharolyticum JW/SL-YS485, a thermophilic anaerobic bacterium under consideration as a biological catalyst for the conversion of cellulosic biomass to ethanol. Based on enzymatic assays and genome sequence analyses, pathways were identified that would lead to the generation of all major products from xylose fermentation: lactate, acetate, ethanol, hydrogen, and carbon dioxide. Pyruvate ferredoxin oxidoreductase is the primary pyruvate decarboxylating enzyme, producing carbon dioxide, reduced ferredoxin, and acetyl-CoA, and ferredoxin is likely oxidized by a specific hydrogenase. It is concluded that enzymes are present in this organism that could theoretically produce ethanol from carbohydrates at high yield.
    Enzyme and Microbial Technology 05/2008; 42(6-42):453-458. DOI:10.1016/j.enzmictec.2008.01.005 · 2.97 Impact Factor
  • Francis E Jenney, Michael W W Adams
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    ABSTRACT: Hydrogenases are enzymes found in all domains of life that catalyze a remarkably simple chemistry, the reversible oxidation of molecular hydrogen to protons and electrons. In order to perform this chemistry, cells have evolved, several different times, intricate organometal complexes built around a binuclear Ni-Fe or Fe-Fe center, with bound CO and CN(-) groups, as well as multiple FeS centers. These complicated enzymes have been an area of intense study for many decades, with interest peaking on the occasions of major increases in national energy costs. Interest in biologically generated hydrogen as a potential substitute for fossil fuels is again at the forefront, and the new tools of the postgenomic world available for manipulating these enzymes make it a truly viable possibility. Hydrogenases from hyperthermophilic microorganisms such as Pyrococcus furiosus and Thermotoga maritima, with optimal growth temperatures near 100 degrees C, are of particular interest and promise for elucidating and manipulating these enzymatic mechanisms.
    Annals of the New York Academy of Sciences 04/2008; 1125:252-66. DOI:10.1196/annals.1419.013 · 4.31 Impact Factor

Publication Stats

2k Citations
417.88 Total Impact Points


  • 2009–2013
    • Philadelphia College of Osteopathic Medicine
      Philadelphia, Pennsylvania, United States
  • 1999–2011
    • University of Georgia
      • • Department of Biochemistry and Molecular Biology
      • • Complex Carbohydrate Research Center
      • • Department of Chemistry
      • • Center for Metalloenzyme Studies
      Атина, Georgia, United States
  • 2007
    • Chinese Academy of Sciences
      Peping, Beijing, China
  • 2005–2006
    • North Carolina State University
      • • Department of Chemical and Biomolecular Engineering
      • • Department of Microbiology
      Raleigh, North Carolina, United States
    • Stanford University
      • Department of Chemistry
      Palo Alto, California, United States
  • 2004
    • Los Alamos National Laboratory
      • Bioscience Division
      Los Alamos, California, United States
  • 2003
    • Louisiana State University
      • Department of Chemistry
      Baton Rouge, Louisiana, United States
  • 1993–1997
    • University of Pennsylvania
      • Department of Biology
      Philadelphia, PA, United States
  • 1995
    • University of Bologna
      Bolonia, Emilia-Romagna, Italy