Edward Eisenstein

Loyola University Maryland, Baltimore, Maryland, United States

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Publications (68)212.18 Total impact

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    ABSTRACT: Recepteur d'Origine Nantais (RON1) receptor tyrosine kinase and its ligand, serum Macrophage Stimulating Protein (MSP), play important roles in inflammation, cell growth, migration and epithelial to mesenchymal transition during tumor development. The binding of mature MSPαβ (disulfide-linked α and β chains) to RON ectodomain modulates receptor dimerization, followed by autophosphorylation of tyrosines in the cytoplasmic receptor kinase domains. Receptor recognition is mediated by binding of MSP β-chain (MSPβ) to the RON Sema domain. Here we report the structure of RON Sema-PSI-IPT1 (SPI1) domains in complex with MSPβ at 3.0 Å resolution. The MSPβ serine protease-like β-barrel uses the degenerate serine protease active site to recognize blades 2, 3, and 4 of the β-propeller fold of RON Sema. Despite the sequence homology between RON and MET and between MSP and HGF, it is well established that there is no cross reactivity between the two receptor-ligand systems. Comparison of the structure of RON SPI1 in complex with MSPβ and that of MET Sema-PSI in complex with HGFβ reveals the receptor-ligand selectivity determinants. Analytical ultracentrifugation studies of the SPI1/MSPβ interaction confirm the formation of a 1:1 complex. SPI1 and MSPαβ also associate primarily as a 1:1 complex with a similar binding affinity to that of SPI1/MSPβ. In addition, the SPI1/MSPαβ ultracentrifuge studies reveals a low abundance 2:2 complex with ~10-fold lower binding affinity compared with the 1:1 species. These results support the hypothesis that the α-chain of MSPαβ mediates RON dimerization.
    Journal of Biological Chemistry 09/2014; 289(43). DOI:10.1074/jbc.M114.594341 · 4.60 Impact Factor
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    ABSTRACT: The diversity of useful compounds produced by plant secondary metabolism has stimulated broad systems biology approaches to identify the genes involved in their biosynthesis. Systems biology studies in non-model plants pose interesting but addressable challenges, and have been greatly facilitated by the ability to grow and maintain plants, develop laboratory culture systems, and profile key metabolites in order to identify critical genes involved their biosynthesis. In this chapter we describe a suite of approaches that have been useful in Actaea racemosa (L.; syn. Cimicifuga racemosa, Nutt., black coshosh), a non-model medicinal plant with no genome sequence and little horticultural information available, that have led to the development of initial gene-metabolite relationships for the production of several bioactive metabolites in this multicomponent botanical therapeutic, and that can be readily applied to a wide variety of under-characterized medicinal plants.
    Methods in molecular biology (Clifton, N.J.) 01/2014; 1083:253-73. DOI:10.1007/978-1-62703-661-0_15 · 1.29 Impact Factor
  • Bhavneet Kaur, Joe-Ann McCoy, Edward Eisenstein
    American Journal of Plant Sciences 01/2013; 04(05):77-83. DOI:10.4236/ajps.2013.45A012
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    ABSTRACT: Protein-protein interactions identified through high-throughput proteomics efforts continue to advance our understanding of the protein interactome. In addition to highly specific protein-protein interactions, it is becoming increasingly more common for yeast two-hybrid, pull-down assays, and other proteomics techniques to identify multiple protein ligands that bind to the same target protein. A resulting challenge is to accurately characterize the assembly of these multiprotein complexes and the competition among multiple protein ligands for a given target. The Association of Biomolecular Resource Facilities-Molecular Interactions Research Group recently conducted a benchmark study to assess participants' ability to correctly describe the interactions between two protein ligands and their target protein using primarily biosensor technologies, such as surface plasmon resonance. Participants were provided with microgram quantities of three proteins (A, B, and C) and asked to determine if a ternary A-B-C complex can form or if protein-B and protein-C bind competitively to protein-A. This article will summarize the experimental approaches taken by participants to characterize the molecular interactions, the interpretation of the data, and the results obtained using different biosensor instruments.
    Journal of biomolecular techniques: JBT 09/2012; 23(3):101-14. DOI:10.7171/jbt.12-2303-003
  • Journal of biomolecular techniques: JBT 01/2011; 22(Suppl):S23.
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    ABSTRACT: Black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa, Nutt., Ranunculaceae) is a popular herb used for relieving menopausal discomforts. A variety of secondary metabolites, including triterpenoids, phenolic dimers, and serotonin derivatives have been associated with its biological activity, but the genes and metabolic pathways as well as the tissue distribution of their production in this plant are unknown. A gene discovery effort was initiated in A. racemosa by partial sequencing of cDNA libraries constructed from young leaf, rhizome, and root tissues. In total, 2,066 expressed sequence tags (ESTs) were assembled into 1,590 unique genes (unigenes). Most of the unigenes were predicted to encode primary metabolism genes, but about 70 were identified as putative secondary metabolism genes. Several of these candidates were analyzed further and full-length cDNA and genomic sequences for a putative 2,3 oxidosqualene cyclase (CAS1) and two BAHD-type acyltransferases (ACT1 and HCT1) were obtained. Homology-based PCR screening for the central gene in plant serotonin biosynthesis, tryptophan decarboxylase (TDC), identified two TDC-related sequences in A. racemosa. CAS1, ACT1, and HCT1 were expressed in most plant tissues, whereas expression of TDC genes was detected only sporadically in immature flower heads and some very young leaf tissues. The cDNA libraries described and assorted genes identified provide initial insight into gene content and diversity in black cohosh, and provide tools and resources for detailed investigations of secondary metabolite genes and enzymes in this important medicinal plant.
    Plant Cell Reports 12/2010; 30(4):613-29. DOI:10.1007/s00299-010-0979-5 · 2.94 Impact Factor
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    ABSTRACT: Pseudomonas quinolone signal (PQS), 2-heptyl-3-hydroxy-4-quinolone, is an intercellular alkyl quinolone signaling molecule produced by the opportunistic pathogen Pseudomonas aeruginosa. Alkyl quinolone signaling is an atypical system that, in P. aeruginosa, controls the expression of numerous virulence factors. PQS is synthesized from the tryptophan pathway intermediate, anthranilate, which is derived either from the kynurenine pathway or from an alkyl quinolone specific anthranilate synthase encoded by phnAB. Anthranilate is converted to PQS by the enzymes encoded by the pqsABCDE operon and pqsH. PqsA forms an activated anthraniloyl-CoA thioester that shuttles anthranilate to the PqsD active site where it is transferred to Cys112 of PqsD. In the only biochemically characterized reaction, a condensation then occurs between anthraniloyl-PqsD and malonyl-CoA or malonyl-ACP, a second PqsD substrate, forming 2,4-dihydroxyquinoline (DHQ). The role PqsD plays in the biosynthesis of other alkyl quinolones, such as PQS, is unclear, though it has been reported to be required for their production. No evidence exists that DHQ is a PQS precursor, however. Here we present a structural and biophysical characterization of PqsD that includes several crystal structures of the enzyme, including that of the PqsD-anthranilate covalent intermediate and the inactive Cys112Ala active site mutant in complex with anthranilate. The structure reveals that PqsD is structurally similar to the FabH and chalcone synthase families of fatty acid and polyketide synthases. The crystallographic asymmetric unit contains a PqsD dimer. The PqsD monomer is composed of two nearly identical approximately 170-residue alphabetaalphabetaalpha domains. The structures show anthranilate-liganded Cys112 is positioned deep in the protein interior at the bottom of an approximately 15 A long channel while a second anthraniloyl-CoA molecule is waiting in the cleft leading to the protein surface. Cys112, His257, and Asn287 form the FabH-like catalytic triad of PqsD. The C112A mutant is inactive, although it still reversibly binds anthraniloyl-CoA. The covalent complex between anthranilate and Cys112 clearly illuminates the orientation of key elements of the PqsD catalytic machinery and represents a snapshot of a key point in the catalytic cycle.
    Biochemistry 09/2009; 48(36):8644-55. DOI:10.1021/bi9009055 · 3.19 Impact Factor
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    ABSTRACT: Thioesters play a central role in the cells where they participate in metabolism, membrane synthesis, signal transduction, and gene regulation. Thioesters are converted to the thiol and carboxylic acid components by thioesterase-catalyzed hydrolysis. Here we examine the biochemical and biological function of the hot dog fold thioesterase YciA (EcYciA) from Escherichia coli and its close sequence homologue HI0827 from Haemophilus influenzae (HiYciA). The quaternary structure of HiYciA was determined, using equilibrium sedimentation techniques, to be a homohexamer. Mass spectral and (31)P NMR analysis of purified HiYciA revealed a bound CoA ligand. Kinetic analyses showed that CoA is a strong feedback inhibitor. YciA thioesterase activity toward acyl-CoA substrates was determined using steady-state kinetic methods. The k cat and k cat/ K m values obtained reveal a striking combination of high catalytic efficiency and low substrate specificity. The substrate activity of propionyl-s- N-acetylcysteine was found to be negligible and that of n-butyryl-pantetheinephosphate low, and therefore, it is evident YciA does not target acylated ACPs or other acylated proteins as substrates. The results from bioinformatic analysis of the biological distribution and genome contexts of yciAs are reported. We conclude that YciA is responsible for the efficient, "seemingly" indiscriminant, CoA-regulated hydrolysis of cellular acyl-CoA thioesters in a wide range of bacteria and hypothesize that this activity may support membrane biogenesis.
    Biochemistry 04/2008; 47(9):2789-96. DOI:10.1021/bi702334h · 3.19 Impact Factor
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    ABSTRACT: The solution structure of the 154-residue conserved hypothetical protein HI0004 has been determined using multidimensional heteronuclear NMR spectroscopy. HI0004 has sequence homologs in many organisms ranging from bacteria to humans and is believed to be essential in Haemophilus influenzae, although an exact function has yet to be defined. It has a alpha-beta-alpha sandwich architecture consisting of a central four-stranded beta-sheet with the alpha2-helix packed against one side of the beta-sheet and four alpha-helices (alpha1, alpha3, alpha4, alpha5) on the other side. There is structural homology with the eukaryotic matrix metalloproteases (MMPs), but little sequence similarity except for a conserved region containing three histidines that appears in both the MMPs and throughout the HI0004 family of proteins. The solution structure of HI0004 is compared with the X-ray structure of an Aquifex aeolicus homolog, AQ_1354, which has 36% sequence identity over 148 residues. Despite this level of sequence homology, significant differences exist between the two structures. These differences are described along with possible functional implications of the structures.
    Protein Science 03/2005; 14(2):424-30. DOI:10.1110/ps.041096705 · 2.86 Impact Factor
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    ABSTRACT: PhzG is a flavin-dependent oxidase that is believed to play a role in phenazine antibiotic synthesis in various bacteria, including Pseudomonas. Phenazines are chorismic acid derivatives that provide the producing organisms, including the opportunistic pathogen P. aeruginosa, with a competitive growth advantage. Here, the crystal structures of PhzG from both P. aeruginosa and P. fluorescens solved in an unliganded state at 1.9 and 1.8 A resolution, respectively, are described. Although the specific reaction in phenazine biosynthesis catalyzed by PhzG is unknown, the structural data indicates that PhzG is closely related to pyridoxine-5'-phosphate oxidase, the Escherichia coli pdxH gene product, which catalyzes the final step in pyridoxal-5'-phosphate (PLP) biosynthesis. A previous proposal suggested that the physiological substrate of PhzG to be 2,3-dihydro-3-hydroxyanthranilic acid (DHHA), a phenazine precursor produced by the sequential actions of the PhzE and PhzD enzymes on chorismate, and that two DHHA molecules dimerized in another enzyme-catalyzed reaction to yield phenazine-1-carboxylate. However, it was not possible to demonstrate any in vitro activity upon incubation of PhzG and DHHA. Interestingly, analysis of the in vitro activities of PhzG in combination with PhzF suggests that PhzF acts on DHHA and that PhzG then reacts with a non-aromatic tricyclic phenazine precusor to catalyze an oxidation/aromatization reaction that yields phenazine-1-carboxylate. It is proposed that phzG arose by duplication of pdxH and that the subtle differences seen between the structures of PhzG and PdxH correlate with the loss of the ability of PhzG to catalyze PLP formation. Sequence alignments and superimpositions of the active sites of PhzG and PdxH reveal that the residues that form a positively charged pocket around the phosphate of PLP in the PdxH-PLP complex are not conserved in PhzG, consistent with the inability of phosphorylated compounds to serve as substrates for PhzG.
    Acta Crystallographica Section D Biological Crystallography 12/2004; 60(Pt 11):2110-3. DOI:10.1107/S0907444904022474 · 7.23 Impact Factor
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    ABSTRACT: Phenazines, including pyocyanin and iodonin, are biologically active compounds that are believed to confer producing organisms with a competitive growth advantage, and also are thought to be virulence factors in certain diseases including cystic fibrosis. The basic, tricyclic phenazine ring system is synthesized in a series of poorly characterized steps by enzymes encoded in a seven-gene cistron in Pseudomonas and other organisms. Despite the biological importance of these compounds, and our understanding of their mode of action, the biochemistry and mechanisms of phenazine biosynthesis are not well resolved. Here we report the 1.8 A crystal structure of PhzF, a key enzyme in phenazine biosynthesis, solved by molecular replacement. PhzF is structurally similar to the lysine biosynthetic enzyme diaminopimelate epimerase, sharing an unusual fold consisting of two nearly identical domains with the active site located in an occluded cleft between the domains. Unlike diaminopimelate epimerase, PhzF is a dimer in solution. The two apparently independent active sites open toward opposite sides of the dimer and are occupied by sulfate ions in the structure. In vitro experiments using a mixture of purified PhzF, -A, -B, and -G confirm that phenazine-1-carboxylic acid (PCA) is readily produced from trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) without aid of other cellular factors. PhzA, -B, and -G have no activity toward DHHA. However, in the presence of PhzF, individually or in combinations, they accelerate the formation of PCA from DHHA and therefore appear to function after the action of PhzF. Surprisingly, PhzF is itself capable of producing PCA, albeit slowly, from DHHA. These observations suggest that PhzF catalyzes the initial step in the conversion of DHHA to PCA, probably via a rearrangement reaction yielding the more reactive 3-oxo analogue of DHHA, and that subsequent steps can occur spontaneously. A hypothetical model for how DHHA binds to the PhzF active site suggests that Glu45 and Asp208 could act as general acid-base catalysts in a rearrangement reaction. Given that four reactions lie between DHHA and PCA, ketone formation, ring formation, decarboxylation, and oxidation, we hypothesize that the similar PhzA and -B proteins catalyze ring formation and thus may be more than noncatalytic accessory proteins. PhzG is almost certainly an oxidase and is predicted to catalyze the final oxidation/aromatization reaction.
    Biochemistry 11/2004; 43(39):12427-35. DOI:10.1021/bi049059z · 3.19 Impact Factor
  • Journal of Biomolecular NMR 07/2004; 29(2):213-4. DOI:10.1023/B:JNMR.0000019242.88149.c5 · 3.31 Impact Factor
  • Journal of Biomolecular NMR 06/2004; 29(1):101-2. DOI:10.1023/B:JNMR.0000019469.77977.72 · 3.31 Impact Factor
  • Methods in Enzymology 02/2004; 380:85-106. DOI:10.1016/S0076-6879(04)80004-1 · 2.19 Impact Factor
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    ABSTRACT: Fully characterizing the interactions involving biomolecules requires information on the assembly state, affinity, kinetics, and thermodynamics associated with complex formation. The analytical technologies often used to measure biomolecular interactions include analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). In order to evaluate the capabilities of core facilities to implement these technologies, the Association of Biomolecular Resource Facilities (ABRF) Molecular Interactions Research Group (MIRG) developed a standardized model system and distributed it to a panel of AUC, ITC, and SPR operators. The model system was composed of a well-characterized enzyme-inhibitor pair, namely bovine carbonic anhydrase II (CA II) and 4-carboxybenzenesulfonamide (CBS). Study participants were asked to measure one or more of the following: (1) the molecular mass, homogeneity, and assembly state of CA II by AUC; (2) the affinity and thermodynamics for complex formation by ITC; and (3) the affinity and kinetics of complex formation by SPR. The results from this study provide a benchmark for comparing the capabilities of individual laboratories and for defining the utility of the different instrumentation.
    Journal of biomolecular techniques: JBT 01/2004; 14(4):247-69.
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    ABSTRACT: The bacterial protein encoded by the gene ychF is 1 of 11 universally conserved GTPases and the only one whose function is unknown. The crystal structure determination of YchF was sought to help with the functional assignment of the protein. The YchF protein from Haemophilus influenzae was cloned and expressed, and the crystal structure was determined at 2.4 A resolution. The polypeptide chain is folded into three domains. The N-terminal domain has a mononucleotide binding fold typical for the P-loop NTPases. An 80-residue domain next to it has a pronounced alpha-helical coiled coil. The C-terminal domain features a six-stranded half-barrel that curves around an alpha-helix. The crablike three-domain structure of YchF suggests the binding site for a double-stranded nucleic acid in the cleft between the domains. The structure of the putative GTP-binding site is consistent with the postulated guanine specificity of the protein. Fluorescence measurements have demonstrated the ability of YchF to bind a double-stranded nucleic acid and GTP. Taken together with other experimental data and genomic analysis, these results suggest that YchF may be part of a nucleoprotein complex and may function as a GTP-dependent translation factor.
    Journal of Bacteriology 08/2003; 185(14):4031-7. DOI:10.1128/JB.185.14.4031-4037.2003 · 2.69 Impact Factor
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    ABSTRACT: PhzD from Pseudomonas aeruginosa is an isochorismatase involved in phenazine biosynthesis. Phenazines are antimicrobial compounds that provide Pseudomonas with a competitive advantage in certain environments and may be partly responsible for the persistence of Pseudomonas infections. In vivo, PhzD catalyzes the hydrolysis of the vinyl ether functional group of 2-amino-2-deoxyisochorismate, yielding pyruvate and trans-2,3-dihydro-3-hydroxyanthranilic acid, which is then utilized in the phenazine biosynthetic pathway. PhzD also catalyzes hydrolysis of the related vinyl ethers isochorismate, chorismate, and 4-amino-4-deoxychorismate. Here we report the 1.5 A crystal structure of native PhzD, and the 1.6 A structure of the inactive D38A variant in complex with isochorismate. The structures reveal that isochorismate binds to the PhzD active site in a trans-diaxial conformation, and superposition of the structures indicates that the methylene pyruvyl carbon of isochorismate is adjacent to the side chain carboxylate of aspartate 38. The proximity of aspartate 38 to isochorismate and the complete loss of activity resulting from the conversion of aspartate 38 to alanine suggest a mechanism in which the carboxylate acts as a general acid to protonate the substrate, yielding a carbocation/oxocarbonium ion that is then rapidly hydrated to form a hemiketal intermediate, which then decomposes spontaneously to products. The structure of PhzD is remarkably similar to other structures from a subfamily of alpha/beta-hydrolase enzymes that includes pyrazinamidase and N-carbamoylsarcosine amidohydrolase. However, PhzD catalyzes unrelated chemistry and lacks a nucleophilic cysteine found in its close structural relatives. The vinyl ether hydrolysis catalyzed by PhzD represents yet another example of the catalytic diversity seen in the alpha/beta-hydrolase family, whose members are also known to hydrolyze amides, phosphates, phosphonates, epoxides, and C-X bonds.
    Biochemistry 06/2003; 42(19):5684-93. DOI:10.1021/bi027385d · 3.19 Impact Factor
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    ABSTRACT: D-Tyr-tRNA(Tyr) deacylase is an editing enzyme that removes d-tyrosine and other d-amino acids from charged tRNAs, thereby preventing incorrect incorporation of d-amino acids into proteins. A model for the catalytic mechanism of this enzyme is proposed based on the crystal structure of the enzyme from Haemophilus influenzae determined at a 1.64-A resolution. Structural comparison of this dimeric enzyme with the very similar structure of the enzyme from Escherichia coli together with sequence analyses indicate that the active site is located in the dimer interface within a depression that includes an invariant threonine residue, Thr-80. The active site contains an oxyanion hole formed by the main chain nitrogen atoms of Thr-80 and Phe-79 and the side chain amide group of the invariant Gln-78. The Michaelis complex between the enzyme and D-Tyr-tRNA was modeled assuming a nucleophilic attack on the carbonyl carbon of D-Tyr by the Thr-80 O(gamma) atom and a role for the oxyanion hole in stabilizing the negatively charged tetrahedral transition states. The model is consistent with all of the available data on substrate specificity. Based on this model, we propose a substrate-assisted acylation/deacylation-catalytic mechanism in which the amino group of the D-Tyr is deprotonated and serves as the general base.
    Journal of Biological Chemistry 05/2003; 278(15):13496-502. DOI:10.1074/jbc.M213150200 · 4.60 Impact Factor
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    ABSTRACT: Small-angle neutron scattering and contrast variation were used to study the solution structure of GroEL and GroEL/GroES chaperonins complexed with a nonnative variant of the polypeptide substrate, subtilisin (PJ9). The subtilisin was 86% deuterated (dPJ9) so that it contrasted sufficiently with the chaperonin, allowing the contrast variation technique to be used to separate the scattering from the two components bound in the complex. Both the native double-ring GroEL and a single-ring mutant were used with dPJ9 bound in a 1:1 stoichiometry per GroEL toroid. This allowed both the position and the shape of dPJ9 in the GroEL/dPJ9 complexes to be determined. A single-ring GroEL/GroES variant complexed with one dPJ9 molecule was used to study the structural changes of dPJ9 in GroEL/GroES/dPJ9 complexes formed with ADP and with ATP. It was found that both the shape and the position of the bound dPJ9 in the GroEL/GroES/dPJ9 complex with ADP were the same as those in the GroEL/dPJ9 complex. However, dPJ9 assumed a more symmetric shape when bound in the GroEL/GroES/dPJ9 complex with ATP. This important observation reflects the relative ability of ATP to promote refolding of protein substrates relative to that of ADP.
    Journal of Structural Biology 04/2003; 141(3):240-58. DOI:10.1016/S1047-8477(03)00002-9 · 3.37 Impact Factor
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    ABSTRACT: The crystal structures of YibK from Haemophilus influenzae (HI0766) have been determined with and without bound cofactor product S-adenosylhomocysteine (AdoHcy) at 1.7 and 2.0 A resolution, respectively. The molecule adopts an alpha/beta fold, with a topology that differs from that of the classical methyltransferases. Most notably, HI0766 contains a striking knot that forms the binding crevice for the cofactor. The knot formation is correlated with an alternative arrangement of the secondary structure units compared with the classical methyltransferases. Two loop regions undergo conformational changes upon AdoHcy binding. In contrast to the extended conformation of the cofactor seen in the classical methyltransferase structures, AdoHcy binds to HI0766 in a bent conformation. HI0766 and its close sequence relatives are all shorter versions of the more remotely related rRNA/tRNA methyltransferases of the spoU sequence family. We propose that the spoU sequence family contains the same core domain for cofactor binding as HI0766 but has an additional domain for substrate binding. The substrate-binding domain is absent in HI0766 sequence family and may be provided by another Haemophilus influenzae partner protein, which is yet to be identified.
    Proteins Structure Function and Bioinformatics 04/2003; 51(1):56-67. DOI:10.1002/prot.10323 · 2.92 Impact Factor

Publication Stats

2k Citations
212.18 Total Impact Points

Institutions

  • 2004–2014
    • Loyola University Maryland
      Baltimore, Maryland, United States
    • Pfizer Inc.
      New York, New York, United States
  • 1993–2014
    • Biomedical Research Institute, Rockville
      Maryland, United States
  • 1994–2004
    • National Institute of Standards and Technology
      • • Biochemical Science Division
      • • Security Technologies Group
      Maryland, United States
  • 1995–2003
    • University of Maryland, Baltimore County
      • Department of Chemistry and Biochemistry
      Baltimore, MD, United States
  • 1998
    • Yale University
      New Haven, Connecticut, United States
  • 1993–1996
    • The University of Tennessee Medical Center at Knoxville
      Knoxville, Tennessee, United States
  • 1994–1995
    • University of Maryland, Baltimore
      Baltimore, Maryland, United States