Chemistry-Biology Interface Symposium Frontiers at the

4th Annual
Frontiers at the
Chemistry-Biology
Interface
Symposium
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! !
The University of Delaware
Saturday April 30th, 2011
8:20-9:00
Registration & Coffee
poster presenters (odd and even numbered) should set up posters.
9:00-9:05
Welcome and opening remarks.
Session I.
Session Chair: Catherine Grimes, U. Delaware
9:05-9:25
James Petersson, Assistant Professor, University of Pennsylvania, Dept. of Chemistry.
Thioamides as Fluorescence Quenching Probes: Minimalist Chromophores for Monitoring
Protein Dynamics
9:25-9:45
Pumtiwitt McCarthy, CBER, U.S. Food and Drug Administration
Preparation of a meningococcal group C polysialic acid- tetanus Hc fragment glycoconjugate
vaccine candidate by chemoenzymatic synthesis
9:45-10:05
Elsa D. Garcin, Assistant Professor, Dept. Chemistry & Biochemistry , UMBC
Down the nitric oxide signaling pathway: structural studies of soluble guanylate cyclase
10:05-10:30
Coffee break
Session II.
Session Chair: Caren Meyers, Asst. Prof. JHU
10:30-10:50
Yaming Hou, Professor, Thomas Jefferson University.
Differentiating analogous tRNA methyltransferases by Analogs of the
Methyl Donor S-adenosyl Methionine.
10:50-11:10
Kory Blocker, Department of Chemical Engineering, University of Delaware,
Tunability and Tailorability of Cell-Triggered DNA Release from a
Substrate-Mediated Delivery System
11:10-11:35
Shuwei Li, Assistant Professor, Dept. Chemistry and Biochemistry, Univ. of Maryland.
Reviving Deuterium-Labeled Tags for Quantitative Proteomics
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1
11:35 1:10
Poster Session I & Lunch
(even numbered posters)
Session III.
Session Chair: Elsa Garcin, Asst. Prof. UMBC
1:10-1:40
Lynn Hyde, Principal Scientist, Merck Research Labs.
Discovery of a Novel Class of Iminoheterocycle BACE1 (b-secretase) Inhibitors With Central
Nervous System (CNS) Activity for the Treatment of Alzheimer’s Disease
1:40-2:00
Jun Wang, Department of Chemistry, University of Pennsylvania
Structure-Based Design Of Inhibitors Targeting Influenza A Virus M2 Proton Channel (A/M2)
2:00-2:30
Pam Green, Professor, University of Delaware, Dept. of Plant and Soil Sciences.
The regulatory world of RNA: Genome-wide analysis of microRNAs and their targets
2:30-3:45
3:45-4:15
Poster Session II /refreshments
(odd numbered posters)
Session IV.
Session Chair:Ed. Lymann, Asst. Prof. UD
James Stivers, Professor, Pharmacology and Molecular Science, Johns Hopkins Univ.
A Look on the Dark Side of Fragment-Based Drug Design
4:15-4:45
Thomas Stevensson, DuPont Crop Protection,
Chemistry at the Interface of Agriculture and Medicine: Pyrazinones as Tubulin Modulators
4:45- 4:50
Closing remarks.
We thank the following for their support:
National Institutes of Health (NIGMS)
The Delaware Biotechnology Institute
The College of Arts and Sciences, University of Delaware
The Johns Hopkins Pharmacology Department
Johns Hopkins Department of Chemistry
University of Maryland, College Park, Department of Chemistry & Biochemistry
University of Maryland Baltimore County, Dept. Chemistry & Biochemistry
The Department of Chemical Engineering, University of Delaware
The Department of Materials Science and Engineering, University of Delaware
The Department of Chemistry and Biochemistry, University of Delaware
Organizing Committee: John Koh, Jennifer Codding and Millicent Sullivan (University of Delaware)
Craig Townsend and Jin Zhang (Johns Hopkins University), Jim Fishbein (University of Maryland,
Baltimore County), Steve Rokita (University of Maryland)
!
2
POSTERS
Poster Session I = even numbered / Poster Session II = odd numbered
Name
1 Mr. Linqing Li
2 Mr. Matthew Weitzman
3 Ms. Franziska Seeger
4 Dr.
Andrew Napper
5 Dr.
Iwona Weidlich
6 Ms. Qin Liang
7 Ms. Sarah Zimmermann
8 Mr. Stephen Kirby
Affiliation
Abstract
University of Delaware A Resilin-based Bio-elastomer for Engineering Mechanically Active Tissues Elastomeric proteins are well known for their large extensibility before
rupture, reversible deformation without loss of energy, and high resilience upon stretching. Motivated by these unique mechanical properties, we are
particularly interested in resilin, the highly elastomeric protein found in specialized compartments of most arthropods; resilin possesses superior resilience and
excellent high-frequency responsiveness. Enabled by biosynthetic strategies, we have designed and expressed recombinant resilin-like polypeptides (RLPs)
bearing both mechanical repetitive domains and biological cassettes. These approaches should allow for the engineering of microenvironments for the
regeneration of mechanically active tissues such as blood vessels, cardiovascular tissues and vocal folds. The cross-linked bio-elastomeric hydrogels display
tunable mechanical stability, efficient energy storage, high resilience, and exhibit high-frequ
University of Delaware Differentiating Highly Conserved Isoforms of the Rho GTPase Subfamily: Rho GTPases are dynamic members of the Ras superfamily, which have been
highly conserved throughout metazoan evolution. Rho GTPases have been implicated in numerous cellular functions, are associated with diverse signaling
networks, and directly interact with an astounding variety of proteins. RhoA and RhoC, members of the Rho subfamily, share 92% sequence identity. Despite
such high homology it is now accepted that each isoform carries out distinct biological functions. We hypothesize that isoform specific functionality is achieved
through non-conserved sites of post-translational modification, differential cellular localization, and discrete membrane association characteristics. In order to
identify the spatiotemporal dynamics of each isoform, we have utilized fluorescently labeled mutant constructs in combination with live cell imaging.
Preliminary results have shown differences in the subcellular localization on each isoform in HE
University of Maryland Biochemical and crystallographic characterization of the regulation and catalysis of soluble Guanylate Cyclase Soluble guanylate cyclase (sGC) is
of Baltimore County
the key enzyme in the NO-sGC-cGMP signaling cascade crucial in regulating the cardiovascular system. NO binding to sGC’s regulatory domain enhances its
basal catalytic activity to convert GTP to cGMP, which in turn modulates downstream targets leading to vasodilation. Low output of this system causes
hypertension and acute heart failure, which are the leading causes of death globally. sGC is a heterodimer. Each of the two homologous subunits (! and ")
contains three domains: an N-terminal regulatory domain (HNOX: Heme Nitric oxide OXygen), a central dimerization HNOX associated (HNOXA) and coiledcoil (CC) domain, and a C-terminal catalytic domain (GC). The enzyme is basally active, but NO binding to the heme group in the " subunit’s regulatory
domain enhances sGC’s catalytic output several hundred fold. The exact mechanism by which the regulatory domain relays t
Nemours Center for
A homogeneous high-throughput screening assay for inhibitors of the interaction of MLL-AF4 and AF9 in pediatric leukemia Translocations of the
Childhood Cancer
mixed lineage leukemia (MLL) gene occur in a subset of aggressive pediatric leukemias. The MLL protein fuses to one of a number of transcription factors,
Research
resulting in potent induction of leukemia through activation of transcription of the oncogene HOXA9, which blocks normal blood cell differentiation. The
transcription factor AF4 is the most common MLL fusion partner, and MLL-AF4 is associated with a particularly poor prognosis. Direct interaction of MLL-AF4
with the transcription factor AF9 appears to be critical to leukemic transformation. We have developed a homogeneous high-throughput screening (HTS)
assay for inhibitors of the binding of MLL-AF4 to AF9 and validated this assay in pilot HTS. Others have shown that peptides derived from AF4 inhibit this
interaction with low nanomolar potency and selectively kill MLL-AF4-containing leukemia cell lines. Our assay m
UMBC
Exploring the Ligand-Based Virtual Screening Study for the Hepatitis C Virus NS5B Polymerase by Docking and QSAR Analysis Hepatitis C virus
(HCV) affects about 170 million people worldwide. Due to the large number of people infected, HCV is an important public health problem. The non structural
NS5B gene product is the RNA dependent RNA polymerase of HCV, which is essential for viral RNA replication. We study this typical right hand structure,
with three distinct Thumb, Fingers and Palm domains. 464 protein-ligand complexes of HCV as well as all related ligands have been downloaded from RCSB
Protein Data Bank and Ligand Expo , respectively. The ligands were clustered into 105 clusters and used as a query for pharmacophore development. The
pharmacophore features of known HCV inhibitors were used as a filter to screen the ChemNavigator iResearch Library of about 59.3 million purchasable
samples, PubChem, and NCI database. Docking of the known inhibitors and hits obtained from virtual screening was perf
University of Delaware Small-molecule inhibitor of a human deubiquitylating enzyme complex. Abstract:Ubiquitin specific proteases (USPs) have been the subject of increased
attention lately as a possible new therapeutic target class. Despite the identification of over sixty USPs in humans and the implication of many USPs in
various human diseases, few small- molecule inhibitors of USPs have been reported. In this work we identified small-molecule inhibitors against the human
USP1/UAF1, a heterodimeric deubiquitinating enzyme complex, through quantitative high throughput screening (qHTS) of a collection of bioactive molecules.
And we also provided the first successful case of targeting the USP-WD40 repeat protein complexes for inhibitor discovery.
UMBC
A series of carbocyclic nucleosides have been designed with the potential of overcoming active site mutations which is one of the leading causes of drug
resistance. These nucleosides have been designed with a flexible purine base motif in which the imidazole and pyrimidine have been split by a single carboncarbon bond, allowing for flexibility to adapt to the active site while still entertaining important hydrogen bonding elements. Recently, we have designed a
series of 5-substituted pyrimidines, “reverse” fleximers, to explore recognition in both purine and pyrimidine metabolizing enzymes. Interestingly, these
compounds have the ability of inhibiting adenosine deaminase (ADA), an important enzyme required for purine metabolism. The synthesis, computational and
enzymatic studies will further be discussed herein.
University of Delaware/ A Mutant of Platelet Activating Factor Acetylhydrolase Hydrolyzes Organophosphorus Nerve Agents Stephen Kirby, Joseph Norris, Douglas Cerasoli
U.S. Army
and Brian Bahnson U.S. Army Medical Research Institute of Chemical Defense, APG, MD 21010. University of Delaware, Department of Chemistry &
Biochemistry, Newark, DE 19716. Organophosphorus compounds (OPs) such as sarin (GB) and soman (GD) are the most toxic chemicals created. They
exert toxic effects by inactivating acetylcholinesterase and binding to secondary targets. OP inhibitors are hemi-substrates for enzymes of the serine
hydrolase superfamily; these enzymes have the potential to be engineered by point mutations into OP-hydrolyzing platforms that can be used as catalytic
bioscavengers. Enzymes associated with lipoproteins, such as human plasma platelet activating factor acetylhydrolase (PAF-AH), have largely been ignored
for engineering purposes due to complex interfacial kinetics and lack of structural data. We have expressed active PAF-AH in bacteria and crystallized the
enzyme-OP adducts. Based on these structures, we engineered histidine mutations (L153H, W298H, F322H) near the permissive active site to enhance
removal of the OP from the phosphorylated serine. Wild-type PAF-AH, L153H, and F322H have essentially no activity against GD, yielding nearly identical
rates from negative control transformations. Surprisingly, W298H displayed novel somanase activity with a kcat of 5 min-1 and a KM of 593#M at pH 7.5.
There was no selective preference for hydrolysis for any of the four GD stereoisomers. Not since the G117H variant of human butyrylcholinesterase has novel
OP-hydrolase activity been engineered into an enzyme via a single amino acid substitution.
9 Mr. Jacob Neal
UMBC
George Washington
University
11 Mr. Vincent Pagnotti
University of the
Sciences
12 Mr. Yubin Bai
Chemistry Dept,
UPenn
13 Ms. Jasmina Cheung-Lau UPenn
10 Dr.
Michael Massiah
14 Mr. Jung Choi
Department of
Chemical Engineering,
University of Delaware
15 Mr. William Bozza
University of Delaware
16 Dr.
University of Maryland
Baltimore
Christopher Baker
17 Ms. Gail Clements
The George
Washington University
Structural characterization of the glyceraldehyde-3-phosphate dehydrogenase-mediated regulation of endothelin-1 expression Glyceraldehyde-3phosphate dehydrogenase (GAPDH) plays a key role in the regulatory pathways of endothelin-1 (ET-1), a vasoconstrictor critical for vascular homeostasis.
Elevated ET-1 levels are associated with certain vascular diseases, thus production and secretion of ET-1 must be tightly regulated. This regulation occurs
primarily via GAPDH-mediated destabilization of ET-1 mRNA. Recent studies suggest that GAPDH binds particular adenine-uridine rich elements (AREs) in
the 3’-untranslated region (3’-UTR) of the ET-1 gene. However, the structural details underlying the GAPDH-mediated control of ET-1 expression are lacking.
With the techniques of molecular biology, electrophoretic mobility shift assay (EMSA), and x-ray crystallography, we seek to probe the structural interaction
between GAPDH and the 3’-UTR of ET-1. To this end, we cloned, expressed, and purified human GAPDH. To identify the specific GAPDH binding
sequences, we constructed small RNA transcripts of the implicated AREs using T7 RNA polymerase. We will next utilize EMSA to identify the smallest RNA
fragments that bind to GAPDH. Ultimately, we will co-crystallize these RNA fragments with GAPDH and solve the structure of the complexes. These studies
will provide the first molecular basis for structure-based drug design, leading to the development of novel molecules that specifically target ET-1 mRNA
destabilization.
Insights into the intrinsically unstructured C-terminus of alpha4, regulator of MID1 and PP2Ac.
A Novel, Ultrasensitive Mass Spectrometry Technique for Sub-pM Detection of Peptides and Proteins. Withdrawn
Hyperpolarized 129Xe chemical exchange saturation transfer (129Xe Hyper-CEST) NMR for ultra high sensitivity detection of xenon host molecules
Self-assembly of thermophilic ferritin towards the encapsulation of gold nanoparticles Abstract: Previously, the incorporation of 10 nm gold
nanoparticles (AuNPs) into the hollow interior of ferritin, taken from the archeon, Archaeoglobus fulgidus, proved successful, as characterized by Joe Swift et
al. (Langmuir 2009). At the conclusion of this study, we determined that the 10 nm AuNPs expand the native framework of the protein. Our current work
involves further characterization of the ferritin-like NP system, synthesized with 5 nm AuNPs using methods that promote ferritin native assembly. The goals
of this project include both the understanding of the bio-inorganic interface, and the development of a stable protein-metal NP with potential application as an
in vivo biosensor. We expand upon earlier work by testing the enzymatic activity of ferritin when annealed to AuNPs, employing electrophoretic assays to
study the stability of ferritin-like particle (FLP) systems, and functionalizing the FLP systems for organization into higher order assemblies.
APPLICATION OF TANDEM MASS SPECTROMETRY FOR QUANTIFYING LABELING DISTRIBUTIONS AND ESTIMATING METABOLIC FLUXES We
have developed and introduced novel methodologies for MFA based on tandem MS and stable-isotope labeling experiments. We demonstrate that tandem
MS provides more labeling information than can be obtained from traditional full scan MS analysis and allows estimation of fluxes with much higher precision.
We present a general modeling framework that takes full advantage of the additional labeling information obtained from tandem MS for MFA. In this
framework, labeling information is obtained from daughter ion scanning and is represented in a matrix form, termed the tandem mass isotopomer matrix. We
show that tandem mass isotopomer matrix can be computed for any compound and any fragmentation using linear mapping of isotopomer fractions. The
inherent advantages of tandem MS compared to full scan MS were demonstrated by comparing the number of independent measurements. For molecules
with more than three atoms tandem MS always provided greater number of independent measurements. To investigate the relationship between the type of
isotopomer measurement and flux identifiability, we generated a series of simulated data sets for a TCA cycle model. The results showed that tandem MS
improved not only the sensitivity of measurements, but also the observability of estimated fluxes. In addition, we developed optimized tandem MS protocols
for accurate and precise measurement of tandem mass isotopomers for TBDMS derivatized amino acids and organic acids. We provide a library of
acceptable fragments that can be used for MFA.
A Peptide-based Multivalent Antagonist of Human Proliferating Cell Nuclear Antigen Proliferating cell nuclear antigen (PCNA) is a master regulator of
cell proliferation via its interactions with numerous proteins, including replicative and specialized DNA polymerases. The natural tumor suppressor, p21,
functions by binding to PCNA and thereby preventing other cellular proteins from accessing PCNA. Targeting this essential interaction, we have designed
tandem repeats of the p21 protein. Our initial hypothesis was a multivalent p21 interaction would demonstrate improved affinity for PCNA than a single p21
peptide. Using isothermal titration calorimetery (ITC) we have confirmed the ability of our divalent p21 in binding PCNA. We have also developed a
fluorescence polarization based competitive peptide binding assay to assess PCNA’s affinity for different p21 peptides. Our results showed that the tandem
p21 repeats have a much improved affinity for PCNA than wild type p21. Our study validated the approach of designing multivalent antagonists of PCNA for
controlling cell proliferation and will facilitate the development of highly potent disruptors of the essential protein interaction with PCNA.
From small molecules to macromolecules: progress towards a CHARMM Drude polarizable force field for the nucleic acids Christopher M. Baker,
Alexander D. MacKerell Jr. Electrostatic interactions play a crucial role in determining the structure and function of biomolecules, and an important aspect of
the electrostatic interaction is polarizability, the response of the molecular dipoles to an external electric field. Towards development of a comprehensive force
field for biomolecules, initial work on the CHARMM Drude polarizable force field for nucleic acids focused on optimizing the theoretical model and developing
parameters for small molecule analogs of the sugar, phosphate and base moieties. With parameters for the small molecule analogs now in place, current
work is focused on construction of the full nucleic acids, and details of this work will be presented here. Initial simulations of the full nucleic acids have
demonstrated that the polarizable model is both robust and stable, and work is now underway to assemble the small molecule building blocks into the full
nucleic acids. This procedure requires careful optimization of the parameters associated with covalent connections between the constituent moieties.
Following initial optimization of the intramolecular connections, multiple simulations will be performed to provide a detailed assessment of macromolecular
properties in comparison to experimental data. These results will be used to identify and correct any weaknesses in the force field, and are also an essential
tool for validation of the model in condensed phase environments; they will give new insights into the importance of polarizability in nucleic acid simulations.
Protein degradation is an essential cellular process. Disruption of protein degradation interferes with numerous cellular activities. Analogous multistep
pathways to degrade proteins are common amongst all living cells. The proteasome, a catalytic enzyme complex, is a major component of proteolysis. Crystal
structures of eukaryotic and an increasing number of prokaryotic proteasomes provide information on their composition and enzymatic activities. Proteasome
inhibitors, already approved for use against cancers, might be significant additions to antibacterial therapies. Our work uses the crystal structure of the
Mycobacterium tuberculosis (Mtb) proteasome as the basis for inhibitor design. Initial rounds of modeling and synthesis are based on analogues of
bortezomib, a boronic acid dipeptide. Current synthetic efforts replace the leucine moiety of bortezomib with substituents that should provide a better fit into
the protein active site, increasing inhibition of the Mtb enzyme. Modeling results, synthesis and biological activity of these new compounds are presented. .
18 Mr. John Larsen
19 Ms. Amy Guminski
20 Mr. Abulfazl Fakhari M.
21 Mr. Kyle Doolan
22 Dr.
Gholson Lyon
23 Mr. Sean Miller
University of Delaware Using the epigenetic code to promote unpackaging from a non-viral, gene delivery vector Abstract: Non-viral gene delivery biomaterials must be able
to release DNA inside the nucleus of a cell. Unfortunately, the majority of non-viral vehicles unpackage prior to nuclear entry or do not unpackage at all,
promoting either premature degradation or inefficient transcription. Thus, we have created DNA polyplexes that are designed to release DNA upon arrival in
the nucleus. These vehicles mimic the natural histone biochemistry that promotes chromosomal DNA activation, and consist of H3 histone tail peptides that
are trimethylated at the fourth position (H3K4Me3) and grafted onto a PEG backbone. The H3K4Me3 modification has been associated with actively
transcribing DNA in a variety of eukaryotes. When the H3K4Me3 peptide is mixed with pDNA, kinetically trapped polyplexes are formed with a hydrodynamic
diameter of approximately 100 nm. We have explored the effects of the N:P packaging ratio on DNA protection and transfection efficiency. Furthermore, upon
insertion of the H3K4Me3 polyplexes into the nucleus, we observe protein production approximately 50% faster than with polyplexes containing
nontrimethylated H3 peptides or with conventional gene delivery materials.
Johns Hopkins
Pyrimidine Nucleotide Imbalances and Innate Immunity to Retroviral Infection Amy F Guminski1, Robert F Siliciano2, James T Stivers1 1Johns Hopkins
University
School of Medicine, Pharmacology and Molecular Sciences and 2Department of Medicine, Baltimore, MD, 21205 Uracil can be found in DNA after either
deamination of cytosine or misincorporation of the deoxynucleotide dUTP during DNA synthesis. Recent work suggests that host organisms may utilize the
detrimental effects of uracil incorporation as a part of their innate immune response. It is suggested that dUTP misincorporation may act as one particular
defense mechanism, as evidenced by the fact that several genera of retroviruses encode a dUTPase to hydrolyze cellular dUTP and reduce dUTP
misincorporation. In these viruses, the encoded dUTPase is essential to permit infection of non-dividing cells, which have a naturally high dUTP:TTP ratio.
Primate lentiviruses such as HIV-1, however, do not encode a dUTPase, which may potentially increase their susceptibility to uracil misincorporation and its
associated detrimental effects. We seek to determine if a high dUTP:TTP ratio can act as an innate barrier to retroviral infection. Using the drug Raltitrexed
(RTX), which inhibits a key step in the de novo pyrimidine biosynthesis pathway, we have been able to greatly increase the dUTP:TTP ratio in HT29 cells.
After RTX treatment, the HIV-1 cDNA of infected cells is significantly uracilated. Although the levels of HIV-1 cDNA are relatively unperturbed by RTX
treatment, the level of integrated provirus is greatly reduced and viral protein production is virtually eliminated. This restriction seems to require UNG, a
cellular Base Excision Repair (BER) enzyme that excises uracil lesions from DNA. We hypothesize that the excision of uracil by UNG from these overuracilated cDNAs prevents stable integration of an infection competent viral genome.
University of Maryland, Criteria for Increasing Efficiency of Excess Electron Transfer in DNA Abstract: Charge transfer processes in DNA are actively studied in various fields
College Park
ranging from biological chemistry to electronics. While parameters affecting the efficiency of hole transfer have been extensively studied, equivalent
investigations on excess electron transfer (EET) in DNA are not yet well resolved. The roles of charge recombination, as a competing process with electron
migration, and the diffusional freedom of electrons during EET in DNA are reported. Charge recombination is reduced using selective electron donation to
pyrimidines by 1-aminoanthracene (AA) as electron donor, in contrast to 1,5-diaminonaphthalene (DAN) that can donate to both purines and pyrimidines.
Results show that AA is about 3-fold more efficient than DAN in inducing EET in identical dsDNA. Studies on a series of dsDNA with a terminal or internal
electron donor reveal that the terminal donor with only one diffusion direction exhibits a 2-fold higher efficiency in transferring electrons in DNA. These findings
may be used in the future to design more efficient EET systems in DNA.
University of Delaware "Hematopoietic Stem Cell Delivery of Therapeutics for the Treatment of Prion Diseases" Kyle M. Doolan and David W. Colby Prion diseases are a
group of rapidly progressing, lethal neurodegenerative diseases for which there currently is no treatment available. Prion diseases are propagated by the
conversion of the native form of the prion protein (PrPC) to an alternatively folded, disease causing form (PrPSc) catalyzed by the interaction of PrPSc with
PrPC. Previous researchers have developed antibodies that bind PrPC and slow the conversion to PrPSc in cell culture and animal models, though few
attempts have been made to engineer existing antibodies for greater therapeutic effect. Beyond protein engineering techniques, delivery of therapeutics using
cell based delivery strategies has the potential to improve therapeutic distribution. Here we propose the development of anti-PrPC single chain antibody
fragments (!-PrPC scFVs) with improved affinity, stability, and endosomal recycling to be delivered by genetically engineered hematopoietic stem cells.
Quantitative evaluation of scFv and cellular parameters will enable the development of a quantitative mathematical model, which will guide future
improvements. The model and methods developed here will likely apply more generally to passive immunity therapies for prion diseases and for cell based
antibody therapies for other neurological diseases.
We identified a family with a previously undescribed lethal X-linked disorder of infancy comprising a unique combination of craniofacial anomalies, hypotonia,
CHOP
global developmental delays, cryptorchidism, cardiac arrhythmia and eventual cardiomyopathy, in which four boys died by approximately one year of age. We
used X-chromosome exome sequencing on an affected newborn individual and his relatives, followed by analysis with a powerful new software program,
VAAST, developed by Mark Yandell at the University of Utah, to identify the causative mutation as c.109T>C; p.S37P in NAA10 (OMIM 300013), a gene that
encodes the catalytic subunit of the major human N-terminal acetyltransferase (NAT). N-terminal acetylation is one of the most common protein modifications
in humans, occurring on approximately 80% of all human proteins, but surprisingly mutations in the NAT genes have never before been linked to human
genetic disease. Functional analysis of hNaa10p S37P compared to wild-type hNaa10p protein demonstrated significantly impaired biochemical activity,
confirming that this mutation causes this disease. More broadly, we have demonstrated that exome sequencing and analysis with VAAST of just one affected
individual, when coupled with pedigree genotype information and follow-up functional assays, can be a powerful technique to identify the mutations that cause
extremely rare Mendelian disorders.
HEDGEHOG GENE REGULATION TO INHIBIT THE NEUROPATHOLOGY OF ALZHEIMER’S DISEASE: IN A TRANSGENIC AD DROSOPHILA MODEL
Drexel University
Alzheimer’s Disease (AD) is the world’s leading cause of dementia with an annual cost of 172 billion dollars. AD is characterized by two hallmark factors:
neurofibrillary tangles (NFT) and amyloid-ß plaques (Aß-plaques). Aß-plaques arise from the aggregation of the Amyloid-Beta (Aß) peptide. Aß is generated
by the sequential cleavage of Amyloid Precursor Protein (APP) by ß and $-secretase enzymes. There is evidence that multiple genes may play a role in the
metabolism of APP and neurodegeneration. Previously, our lab identified Sonic Hedgehog (SHH) as a putative regulator of APP metabolism. SHH plays a key
role in the control of neuronal differentiation, growth, and proliferation. We have established transgenic AD Drosophila melanogaster that express the same
neuropathology as demonstrated by human AD patients. Using this transgenic model we will determine if modulation of the Hedgehog pathway alters the
phenotypes observed in this fly AD model. Determining if SHH plays a role in APP metabolism will further enhance our understanding of AD neuropathology
and may also provide us with possible pharmaceutical interventions.
24 Ms. Fei Li
25 Dr.
Olena Taratula
26 Mr. Woo Suk Ahn
27 Mr. Vidyadhar N.
Daithankar
28 Mrs. Danielle McAtee
29 Dr.
Caren Freel Meyers
30 Mr. Michael McCrane
University of Delaware 77Se NMR spectroscopy of selenoproteins’ redox motifs Selenium, a vital element in the human body, is a cardinal contributor to cellular antioxdative
defense and cancer prevention. Selenoproteins, which contain the reactive amino acid selenocysteine, participate in redox and electron transfer reactions.
The 25 human selenoproteins are powerful enzymes that utilize the selenocysteine in conjunction with a nearby cysteine in their active site. These so called
redox motifs govern the protein redox potential and hence chemical reactivity. We measured the differences in the inherent flexibility of selenocysteine in
different redox motifs: the cysteine-selenocysteine redox motif is present in thioredoxin reductase while the cysteine-X-X-selenocysteine redox motif (X stands
for any amino acid) is found in cell signaling selenoproteins. 77selenocysteine was incorporated into proteins by manipulating E. coli selenium incorporation
machinery. We have measured the relaxation properties of the selenium in the redox motif using solution-state selenium NMR. Our NMR data show that the
selenocysteine in the oxidized cysteine-selenocysteine redox motif is much more rigid than that of the cysteine-X-X-selenocysteine redox motif. In contrast,
the flexibility of the active site selenocysteine is greater in the reduced cysteine-selenocysteine than in the reduced cysteine-X-X-selenocysteine redox motif.
University of
Hyperpolarized 129Xe nuclear magnetic resonance (NMR) biosensors are promising agents for early disease detection since their interactions with target
Pennsylvania
biomolecules can perturb chemical shifts well beyond the typical field inhomogeneity of clinical MRI. We have developed biosensors based on cryptophanes
with the highest reported affinity for xenon, a non-toxic contrast agent. Besides developing an efficient shorter 6-step synthesis for trifunctionalized
cryptophanes, we have also demonstrated the specific cell uptake of cryptophane biosensors modified with specific targeting moieties to bind proteins
upregulated in cancer cells. In addition, human carbonic anhydrase (CA), highly overexpressed in many types of cancer, was chosen as a single-binding-site
enzyme for studying xenon biosensor-protein interactions for bioimaging applications. The development of xenon biosensors capable of generating very large
differences in 129Xe NMR chemical shift between CA isozymes, will allow multiplexing experiments.
Chemical Engineering, Chinese hamster ovary (CHO) cell metabolism at growth and non-growth phase is studied by non-stationary 13C-metabolic flux analysis (MFA)
University of Delaware using [1,2-13C]glucose and gas chromatography-mass spectrometry (GC-MS). As CHO cell is used as major platform to produce bio-therapeutics, it has
key importance to understand the change of cellular metabolic fluxes during cell culture. To study cell metabolism, 13C-MFA is used as a strong tool to
validate biochemical networks and quantify cellular metabolic fluxes. Here, we estimated intracellular fluxes at two phases using non-stationary 13C-MFA with
additional data set external metabolic fluxes as well as mass isotopomer distribution of intracellular metabolites. Through non-stationary 13C-MFA of CHO
metabolism, we observed high glycolytic flux at cell growth phase and high oxidative pentose phosphate activity at non-growth phase. Interestingly, the
pyruvate fluxes to TCA cycle showed similar levels at both phases. TCA cycle fluxes by citrate synthase and pyruvate dehydrogenase were estimated to the
similar levels, but pyruvate cycling fluxes by malic enzyme and pyruvate carboxylase became lower at non-growth phase. As mammalian cell metabolism is
estimated by non-stationary 13C-MFA in cell culture environment, the CHO model and 13C-MFA method would be applied for cell line development by cell
culture engineering and media development to achieve high-titer production of recombinant proteins in biotechnology field.
University of Delaware Sulfhydryl Oxidases Dribble Superoxide Vidyadhar N. Daithankar and Colin Thorpe; Department of Chemistry and Biochemistry, University of Delaware,
Newark, DE 19716 Typical flavoprotein oxidases catalyze the net 2-electron reduction of oxygen with the generation of hydrogen peroxide. While the major
reduced oxygen species directly liberated from the sulfhydryl oxidases (e.g. Erv2p; augmenter of liver regeneration, ALR; and Quiescin-sulfhydryl oxidase,
QSOX), is hydrogen peroxide, about 2-19% of the reduced oxygen species is released directly as the superoxide anion. Superoxide ion flux was detected by
a new kinetic method. During investigations of water-soluble phosphines (e.g. TCEP and THP) as model substrates of the sulfhydryl oxidases, we observed
anomalously high oxygen consumption rates that were drastically suppressed on the addition of superoxide dismutase. Using xanthine/xanthine oxidase, we
then generated a known flux of superoxide ions and measured the consequent acceleration in oxygen consumption in the presence of the phosphine. Our
experiments are consistent with a mechanism in which the superoxide ion initiates a radical chain reaction that sustains the oxidation of phosphines with the
overall stoichiometry: Phosphines + O2 phosphine-oxides + H2O2
Superoxide release by these sulfhydryl oxidases is not confined to phosphine
substrates; it was also confirmed with a luciferin analog, MCLA, using DTT in the presence of Erv2p. The origins, and possible significance, of this
unexpected catalytic activity of the sulfhydryl oxidases will be discussed. More generally, the superoxide dismutase-sensitive amplification of oxygen
consumption encountered in the presence of phosphines can provide an alternative assessment of superoxide release in vitro. Finally, these studies suggest
that caution needs to be exercised when using phosphines as antioxidants in cell cultures. Supported by NIH GM26643
University of Delaware A Nickel(0)-Catalyzed Formal Aza-Nazarov Cyclization for Isoindolinone Synthesis Isoindolinones comprise the core structure of a number of biologically
active compounds. We have discovered that this heterocycle can be easily prepared from readily accessible N-benzoyl aminals using nickel-based catalysts.
Reaction optimization and substrate scope of this reaction is presented, as well as mechanistic studies for this formal aza-Nazarov cyclization.
Johns Hopkins
1-Deoxy-D-xylulose 5-phosphate synthase: A new twist in thiamine diphosphate-dependent enzymology The unique methylerythritol phosphate
University School of
(MEP) pathway for isoprenoid biosynthesis is essential in most human pathogens, including Mycobacterium tuberculosis and the malaria parasite
Medicine
Plasmodium falciparum. The first enzyme in this pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase, catalyzes the thiamin diphosphate (ThDP)dependent formation of DXP from D-glyceraldehyde 3-phosphate (GAP) and pyruvate. This enzyme is mechanistically and structurally distinct from other
ThDP-dependent enzymes and is a potential drug target for the development of new anti-infective agents. Toward our long-term goal to target nonmammalian isoprenoid biosynthesis, we have conducted mechanistic studies of DXP synthase. Here, we present the results of detailed kinetic analysis and
substrate binding studies. Our results support a novel, random sequential mechanism of catalysis for DXP synthase. These results have important
implications for the design of selective inhibitors of isoprenoid biosynthesis.
University of Maryland, “Trapping Reversible ortho-Quinone Methide – Nucleoside Adducts" Electrophilic ortho-quinone methides (o-QM) are generated during xenobiotic
College Park
metabolism of a variety of compounds and can alkylate DNA. From model studies based on nucleosides, o-QMs react most readily, but reversibly with strong
nucleophiles. Their reaction is less efficient, but irreversible with weak nucleophiles. The hour time-scale of the reverse reactions complicates analysis of their
products in DNA, which requires enzymatic digestion and chromatographic separation. Instead, a chemical trap utilizing bis[(trifluoroacetoxy)iodo]benzene
has been developed to transform the reversible o-QM-DNA adducts into irreversible derivatives, capable of surviving such analysis. Preliminary studies,
involving a model o-QM and 2'-deoxycytidine resulted in the isolation of a surprising derivative likely due to over-oxidation. A second model o-QM compound
included an alkyl group to block over-oxidation. A surprising derivative was still generated from oxidation of its adduct, but the final derivative should be
sufficient for identifying labile o-QM adducts in DNA.
31 Mr. Meghan Reilly
32 Mr. Muhammad Noon
33 Mr. Anil Pandey
34 Dr.
Xue Zhi Zhao
35 Ms. Bernice Bediako
University of Delaware Histone-Mimetic Polyplexes as Self-Activating & Tailorable Non-Viral Gene Delivery Vehicles Recent developments with non-viral gene carriers have
focused on enhancing the effectiveness of gene therapies and improving understanding of the cell milieu and endocytic machinery. A novel solution to
address inefficient non-viral DNA delivery is the development of biomimetic materials capable of regulating DNA accessibility and transcriptional activation.
For example, recognition of the histone H3 tail trimethylated at lysine 4 by nucleosome remodeling factors has been implicated in chromatin activation. This
work examines H3 tail peptides both with and without a trimethylated K4 for their potential as gene delivery materials. We have found that the H3 tail peptides,
in combination with the helper cationic polymer poly(ethylenimine) (PEI), bind, protect, and release DNA. Dynamic light scattering and agarose gel
electrophoresis were used to demonstrate that both H3 peptides condense DNA into nanoscale particles that were stable in the presence of serum nucleases.
The H3-PEI hybrid polyplexes were found to transfect a substantially higher number of CHO-K1 cells in vitro compared to both polyplexes that were formed
with only the H3 peptides and those that were formed with only PEI at the same total charge ratio. In addition, the PEI polyplexes exhibited a cytotoxic effect,
whereas the H3-PEI hybrid polyplexes did not compromise cell viability. We have explored the observed synergies between the H3 peptides and PEI through
the use of various chemical endocytic uptake and trafficking inhibitors, and have determined that the endocytic pathway plays a vital role in the fate of the
polyplexes.
School of Pharmacy,
MD simulation of the effect of oxidized methionine on the binding of DNA and CBF! to the Runt domain of CBF" Runt domain (RD) is DNA-binding
University of Maryland, domain of p53 type transcription factors known as core binding factor ! (CBF!) subunits. Three related genes Runx1, Runx2 and Runx3 are involved in the
Baltimore
expression of CBF!s. Runx genes are important for several developmental processes; Runx1 plays role in haematopoiesis, Runx2 is important for skeletal
development (osteogenesis) and Runx3 is essential in the development of gastric epithelium. Mutation of Runx1 is linked with human leukemias, whereas
mutation of Runx3 leads to development and progression of gastric cancers and testicular yolk sac tumor. Runx2 gene mutation is associated with inherited
human skeletal disorder known as cleidocranial dysplasia. Biological activity of some proteins is known to be sensitive to oxidative modifications caused by
variety of oxidants. Methionine, and Cysteine, residues of proteins are particularly sensitive to oxidation by reactive oxygen species (ROS). Methionine
residues may be oxidized to sulfoxide form (reversible) and to sulfone form (permanent change). Here, we carry out molecular dynamic (MD) simulation
studies of RD, which is the DNA-binding region of Runx genes, to see the effect of oxidized (sulfoxide form) methionine-106 of RD on its interactions with its
binding partners. Methionine-106 is located at the interface with a related protein, known as core binding factor " (CBF"), which binds to the RD to enhance
RD-DNA interaction. The simulation studies indicate that oxidation of methionine-106 leads to improved binding of RD with CBF" and DNA as dimers and in
the DNA-RD-CBF" ternary complex.
University of Delaware Proline Editing: A Novel Tool for Controlling Peptide Structure and To Synthesize Chemically Diverse Peptides Abstract: Protein function is
dependent upon protein conformation. Dramatic changes in peptide or protein structure may be achieved through utilization of stereoelectronic effects to
control protein main chain conformation. We have developed a novel chemistry termed Proline Editing, which provides means of rapid modification and
functionalization of a peptide or protein. A model peptide TYXN (X= 4-Hydroxyproline) was used to demonstrate the versatility of this approach by post
synthetic chemical modification at 4 position of proline (on resin) leading to conformational and broad chemical diversity. This easy to do chemistry utilizes a
few washes of the derivatized resin, cleavage and subsequent HPLC purification, which significantly reduce time and material resources. These derivatives
can be utilized as precursors for bio-orthogonal chemistry, as linkers, and steric or electronic based switches. This stereoelectronic approach of tuning the
local structure of peptide and protein via controlling ring pucker of proline and cis-trans isomerization of peptide backbone can be utilized globally to stabilize
protein secondary structures. We have also demonstrated the bioorthogonal application of these post synthetically modified peptides by doing three parallel
reactions together in one vial with excellent selectivity and yields within minutes of the reaction.
NIH
Hydroxy-pyrrolopyridine-trione Based HIV-1 Integrase Inhibitors Abstract: After more than two decades of intensive research, approximately 30 drugs
have been approved by the FDA for the treatment of HIV/AIDS. Merck’s IsentressTM (MK-0518 or Raltegravir), the first FDA-approved HIV-1 integrase (IN)
inhibitor, shares key structural features with other IN inhibitors. These features include a co-planar arrangement of heteroatoms that chelate magnesium ions
and halogen-substituted aromatic functionality linked to the chelating portion of the molecule that interacts with a region formed between a viral DNA base and
the protein in the IN•DNA complex. The emergence of Raltegravir-resistant IN mutants demonstrates a need to develop new IN inhibitors that can overcome
this resistance. A co-crystal of Raltegravir bound to the Prototype Foamy virus (PFV) IN complexed with DNA [Hare, S. et. al. Nature, 2010, 464, 232-7]
provides insights into the basis for the resistance caused by mutations in IN. We have previously reported 4,5-dihydroxy-1H-isoindole-1,3(2H)-diones as
structurally simple IN inhibitors that exhibit good potency and strand transfer selectivity in vitro in the presence of Mg2+ cofactor. The co-crystal structure of
one of these inhibitors bound to the PFV•DNA complex has recently been solved. This allows comparison to be made of the binding interactions of our
synthetic inhibitors with Raltegravir. As reported herein, a series of hydroxy-pyrrolopyridine-trione-containing analogues were prepared by insertion of a
nitrogen into the ring system of the original 4,5-dihydroxy-1H-isoindole-1,3(2H)-diones. This simultaneously combines structural features of our original
inhibitors with Merck’s pyrimidinones IN inhibitors. The efficient synthesis of these compounds relies on the application of a “Pummerer cyclization
deprotonation cycloaddition” cascade of imidosulfoxides as well as [3+2] cycloaddition of isomünchnones. Introducing a nitrogen substituent into the catechol
ring to give the 2(1H)-pyridone moi
University of Maryland Regulation of the GOSPEL-GAPDH-Siah 1 apoptotic pathway by S-nitrosation The GOSPEL-GAPDH-Siah 1 pathway is a recently discovered pathway
Baltimore County
implicated in neurodegenerative diseases. The S-nitrosation of both GOSPEL (GAPDH’s competitor Of Siah Protein Enhances Life) and GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) in the presence of an apoptotic stimulus prevents Siah-1-mediated nuclear translocation of GAPDH, thus
preventing cell death. The main aims of this research are to investigate 1) the effect of nitrosation on the association between GOSPEL and GAPDH and 2)
the effect of nitrosation on GAPDH and GOSPEL 3D structure by combining molecular biology, enzymatic assay, x-ray crystallography and small-angle x-ray
scattering. Our preliminary studies indicate that unmodified GAPDH follows Michealis-Menten kinetics with Vmax, Km and Kcat values of 0.65 #M.s-1, 0.05
mM and 0.013 s-1, respectively. By comparison, CysNO modified GAPDH shows decreased enzymatic catalytic activity (Vmax, Km and Kcat values of 0.16
#M.s-1, 0.08 mM and 0.002 s-1, respectively). Understanding the effects of nitrosation on the apoptotic GOSPEL-GAPDH-Siah 1 pathway may help in the
design of novel therapeutics for the treatment of neurodegenerative diseases.
36 Ms. Francine Morris
37 Ms. Chengyun Huang
38 Mr. Scott Crown
39 Ms. Catherine Grimes
40 Ms. Stephanie Scott
41
Johns Hopkins
University
DXP synthase-catalyzed C-N bond formation: Implications for inhibitor design Authors Francine Morris and Caren Freel Meyers Abstract The rising
occurrence of microbial resistance to all currently available therapies has created a growing demand for new anti-infectives based on novel modes of action.
Several opportunistic pathogens, including Pseudomonas aeruginosa, Mycobacterium tuberculosis and the malaria parasite, Plasmodium falciparum utilize
the methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis. Since the production of isoprenoids is essential and the MEP pathway is absent in
mammals, the enzymes in this pathway are attractive targets for the development of new anti-infective agents. Currently, only a few inhibitors of nonmammalian isoprenoid biosynthesis are known. DXP synthase is the first step in this pathway and is known to catalyze the formation of a C-C bond in the
production of deoxy-D-xylulose 5-phosphate (DXP) from pyruvate and D-glyceraldehyde 3-phosphate. In addition, DXP synthase catalyzes the early steps in
thiamin diphosphate (ThDP) and vitamin B6 biosynthesis. The similarity of this enzyme to the mammalian transketolase superfamily suggests that selective
inhibition of DXP synthase may be a challenging endeavor. However, recent work has demonstrated that DXP synthase possesses a unique catalytic
mechanism and structure, suggesting that development of selective inhibitors is possible. Here, we present a substrate specificity study that highlights DXP
synthase-catalyzed C-N bond formation and reveals a remarkable affinity of the enzyme for naphthol-containing alternative substrates. Current efforts are
focused on studying structure-activity relationships for alternative substrate turnover and identifying important binding elements as a starting point for inhibitor
design.
University of Maryland, Synthesis of an electron rich quinone methide precursor to expedite sequence-directed alkylation of DNA Chengyun Huang, Steven E. Rokita*
College Park
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, 20740 A quinone methide (QM) is a very reactive electrophilic
intermediate that can alkylate the most nucleophilic nitrogens in DNA reversibly. This reversibility has been used to construct sequence-directed alkylating
reagents, but their reaction is slow. We have now synthesized an electron rich quinone methide precursor (QMP) to accelerate QM (re)generation for
selective alkylation. The DNA-QMP conjugate was completely converted to DNA-QM self-adduct within 0.5 hour when the electron donating group was
present compared to a period of 24 hours needed for a parent QM lacking the electron donating group. Alkylation study using the DNA-QM self-adduct with a
single strand DNA target also showed that the electron rich self-adduct needed only half (2 days) as long as the unsubstituted self-adduct (4 days) to reach
maximal alkylation of the target. This enhanced efficiency should shorten the time for a QM to take effect in a biological system.
University of Delaware Novel Insights into Odd-chain Fatty Acid Synthesis in Adipocytes Obesity and type II diabetes are increasing health epidemics in Western society and
are characterized by dysregulation of lipid metabolism. A more complete understanding of de novo lipid synthesis at the metabolic level is required to develop
novel therapeutics for the treatment of these disorders. Towards this end, we are implementing a systems biology approach using metabolic flux analysis and
stable isotope 13C-tracers to determine the active reaction pathways in an adipocyte cell line 3T3-L1. In the work presented here, we investigated the
synthesis of odd-chain length fatty acids in differentiated white adipocytes. The white adipocytes were incubated with [U-13C]-glutamine and incorporation of
13C into fatty acids was analyzed with gas chromatography mass spectrometry (GC-MS). Isotopomer spectral analysis (ISA), conducted on the mass
isotopomer distributions of the fatty acids, suggested that odd-chain fatty acid synthesis proceeded from elongation of a three-carbon precursor rather than by
alpha-oxidation as was previously assumed. To investigate this further, differentiated adipocytes were incubated with [U-13C,15N]-valine, and [U-13C,15N]isoleucine. From these tracer experiments, valine and isoleucine were determined to be major contributors to the three-carbon precursor pool. Furthermore,
novel mass spectrometry techniques validated that the three-carbon moiety acts as the primer on fatty acid synthase and is subsequently elongated by acetylCoA subunits. Lastly, it was verified that the incorporation of three-carbon units is highly specific to odd-chain fatty acids. The research demonstrated here
provides an important link between amino acid and fatty acid metabolism.
Harvard/University of
Chronic inflammatory disorders, such as Inflammatory Bowel Disease, arise from an inappropriate immune response to bacteria. In order to treat
Delaware
these diseases, we need a better understanding of how the innate immune system senses and responds to bacteria. It has long been known that muramyl
dipeptide (MDP), a fragment of bacterial cell wall (peptidoglycan), is able to generate an immune response. Recently, it has been shown that the intracellular,
mammalian protein Nod2 is involved in sensing the presence of MDP in the cell, leading to activation of the synthesis of inflammatory molecules. Nod2 is an
important protein to human health, as mutations in the gene have shown an increased incidence of Crohn’s disease. The mechanism of activation of the Nod2
signaling pathway by MDP is not understood. For example, it is not known if MDP directly interacts with Nod2. We have taken a chemical biological approach
to this problem by synthesizing tagged versions of MDP. This tool was then utilized to probe for an interaction between Nod2 and MDP. We describe the
development of a variety of binding assays and present a model for Nod2 activation via MDP. Our approach has led to a deeper understanding of the
molecular mechanisms used by the innate immune system to sense bacteria.
University of Delaware Exploring the Physiological Function of SMP30 via Enzyme Kinetics and Crystallography Abstract: Senescence marker protein 30 (SMP30), originally
- Bahnson Lab
identified as an anti-aging marker, exhibits a protective effect against apoptosis and oxidative stress and is thought to be indirectly involved in cellular calcium
(Ca2+) homeostasis. Known enzymatic functions of SMP30 include organophosphate hydrolase and lactonase activities. Studies using SMP30 knockout mice
have shown that SMP30 functions as a gulonolactonase in ascorbate biosynthesis in mammals excluding guinea pigs and most primates. However, the amino
acid sequence is highly conserved (70-90%) in vertebrates from humans to frogs, regardless of ability to synthesize ascorbate. The high evolutionary
sequence conservation suggests that the protein has an important biological function in addition to its role in ascorbate biosynthesis. The purpose of this work
is to probe the structure and lactonase activity of human and mouse SMP30 in order to gain insight into the physiological function of the protein. Mouse and
human forms of SMP30 were expressed in E. coli and purified using affinity and ion-exchange chromatography. Lactonase activity of mouse and human
SMP30 toward D-glucono-%-lactone and L-gulono-$-lactone was monitored using a colorimetric assay containing p-nitrophenol. Purified samples were
screened for crystallization using the hanging drop method. Kinetic data showed little difference between the gluconolactonase activities of mouse and human
SMP30. Neither form of SMP30 hydrolyzed L-gulono-$-lactone. Initial crystals of both mouse and human SMP30 were obtained. Prior to obtaining a crystal
structure of the mouse form, a homology model of mouse SMP30 has been made using the recently solved crystal structure of human SMP30 as a template.
42 Mr. Manu Kanwar
43 Dr.
Yalong Zhang
44 Mr. Dong Yang
45 Ms. Hannah Peters
46 Ms. Laura Wang
47 Mr. Shawn Thatcher
Johns Hopkins
University
A universal switch platform based on designed ankyrin-repeat proteins Abstract: A universal protein switch platform that can be easily adapted to bind a
variety of biological targets would greatly facilitate the development of biomolecular sensors for a variety of applications. Designed ankyrin-repeat proteins
(DARPins) are currently recognized as potential antibody alternatives. DARPins have been engineered by others to specifically bind a variety of protein
targets including maltose binding protein (MBP). DARPin based switches hence represent an excellent platform as a recognition motif for the development of
a universal switch platform. We have used a combinatorial approach to create a library of hybrid proteins that are comprised of the MBP-specific DARPin off7
as the input domain and the enzyme TEM1 "-lactamase (BLA) as an output domain of the switch. Specifically, the gene encoding a circularly permuted
variant of BLA was randomly recombined within the off7 gene to create a hybrid DNA library with a theoretical diversity on the order of 105. An in vivo
selection scheme based on a previously developed bacterial band-pass genetic selection system was implemented to identify fusion proteins in which MBP
activated BLA enzyme activity. The identified switches would be used for the subsequent development of biosensors against a variety of targets by
engineering new binding properties into the input domain.
National Cancer
High Rates of Epimerization During the Synthesis of Glycopeptides Yalong Zhang, Saddam M. Muthana, David Farnsworth, Olaf Ludek, Jeffrey C.
Institute, Frederick
Gildersleeve Glycoproteins play an important role in many biological processes including cell-cell recognition, inflammation, and metastasis. Homogenous
glycoproteins or glycopeptides, however, are difficult to obtain or purify from natural sources due to the template-independent glycosylation with
microheterogeneity.Glycopeptides can be obtained via solid phase synthesis using protected amino acids that contain the glycan (glyco-amino acids) as
building blocks. Compared with standard peptides via automated methods, the synthesis of glycopeptides/glycoproteins can be significantly more challenging.
Because of expensive or synthesized building blocks, use of a large excess of glyco-amino acid to increase the rate and drive the reaction to completion is
unappealing. Scientists typically prepare the peptide precursor up to the glycosylation site on an automated synthesizer and then carry out the coupling of the
glyco-amino acid manually. This process is especially disadvantageous when preparing large numbers of glycopeptides, such as in the construction of a
glycopeptide library. In addition, epimerization of glyc-amino acid during coupling is not evaluated, which is critical for developing glycopeptides vaccines. In
this study, we carried out a systematic comparison of published coupling conditions using four different Fmoc-protected glyco-amino acids as well as the nonglycosylated counterpart. Both efficiency and epimerization were evaluated using HPLC assays. Interestingly, epimerization at the alpha position of the amino
acid backbone was found to be a substantial problem in a number of reactions with glyco-amino acids. In fact, epimerization resulted in as much as 80% of
the non-natural epimer, indicating that the non-natural epimer can be the favored product for some glyco-amino acids. To better understand the origin of this
problem, we also measured rates of ...
University of Maryland The two rings in a GroEL molecule operate ~180o out of phase with one another to fold substrate proteins (SP). The release of ADP from the trans ring
College Park
largely determines duration of the chaperonin cycle. A network of salt bridges break and form during the large, rigid body conformational changes in a ring.
Intra-subunit, inter-domain salt bridge D83-K237 and inter-subunit, inter-domain salt bridge R197-E386 break during the T to R transition. Elimination of these
salt bridges by site directed mutagenesis destabilizes the T state, thus favoring the R state of GroEL rings. At high concentration of ATP and K+, this double
mutant GroEL (D83A/R197A) undergoes only TR to RR transition with a significantly decreased ATPase activity. However, at lower concentration of ATP and/
or K+, the TT to TR transition resumes with increased ATPase activity. This is in accordance with our previous conclusion that the off-rate of ADP is the
limiting step in a chaperonin cycle, and that K+ enhances nucleotide affinity greater to the R state than to the T state. The deficiency of SP-stimulated ATPase
activity at physiological concentration of ATP suggests that the trans ring must cycle through the T state, the SP-acceptor state, promoted by the salt bridges
network, to efficiently assist protein folding.
University of Maryland The most prevalent problem for antiviral agents today is the rapid emergence of resistance to currently used therapeutics. As a result, there is a critical
Baltimore County
demand for drugs that can overcome mutations in order to stay active against resistant viral strains. Recent reports that two new drugs, tenofovir and
etravirine, appear to be able to do just that are highly encouraging. They use their inherent flexibility to alter their conformation and position, allowing them to
overcome mutations in HIV’s reverse transcriptase binding site and retain their potency. Based upon these findings our lab has begun work on the design and
synthesis of a series of nucleoside analogs in which flexibility has been introduced by splitting the purine nucleobase. We have applied this approach to
several known nucleoside inhibitors such as tenofovir and the viral targets include the polymerases of HIV, HCV, HBV, HCMV and HSV. It is hypothesized
that by introducing flexibility into already active nucleoside inhibitors they will be able to retain potency against mutant polymerases in which steric or
electronic mutations have been introduced in the nucleoside binding site. The preliminary synthesis is presented herein.
University of
Error-prone nucelotide incorporation by DinB, a Y-family polymerase from drug-resistant Pseudomonas aeruginosa Abstract: Pseudomonas
Pennsylvania
aeruginosa colonizes the dysfunctional lungs of patients with cystic fibrosis. Through the progressive disease course, the isolates of this opportunistic
pathogen can become increasingly drug resistant, often greatly limiting therapeutic options. DinB, a member of the Y-family of DNA polymerases, is part of
the SOS stress response pathway of the pathogen Pseudomonas aeruginosa. DinB promotes error-prone replication of the bacterial genome and as such has
been implicated in hypermutation and consequent drug resistance. Targeting the activity of this polymerase, therefore, could slow the development of
resistance and prolong the useful lifetimes of current antibiotics. Our studies have focused on the kinetics of nucleotide incorporation--and misincorporation-by the polymerase. We are also exploring the hypothesis that the more open active site of Y-family polymerases will make them amenable to inhibition by
nucleoside analogs such as 3TC, commonly used to target the error-prone reverse transcriptase of HIV.
University of Delaware/ MicroRNAs Associated with Environmental Stress in Arabidopsis Thaliana Environmental stress currently contributes to approximately 50% of crop loss
CBI
world-wide, with climate change expected to exacerbate this problem. Genomics studies have indicated that differences in stress tolerance arise not only from
the presence or absence of stress-associated genes but also from their regulation. In plants, miRNAs are key gene regulators that have been shown to
control various developmental processes and also to be involved in response to environmental stresses. In order to further assess the role of miRNAs in
environmental stress, a series of stress treatments was carried out in Arabidopsis thaliana. Comparisons between small RNA populations of different tissues,
mutants and stresses were made via the construction, sequencing and analysis of more than 25 small RNA libraries, resulting in over 150 million sequences.
Through this method, several novel miRNAs were discovered. Many new miRNAs coming from previously annotated miRNA precursors were also found.
Additionally, some miRNAs and their target genes were shown to be regulated in a novel manner under stress conditions. Despite the prevalence of multiple
simultaneous stresses in agriculture, this area remains largely unexplored. To address this, small RNA libraries were also generated from plants subjected to
dual stresses. Several miRNAs were found to be regulated in a unique manner under these conditions, highlighting the importance of further study in this
area.
48 Mr. Nicholas Corsepius
49 Ms. Meagan Small
50 Dr.
Marina Bellani
51 Mr. Christopher Nabel
52 Ms. Jennifer Codding
53 Mr. Jacob Goldberg
54 Ms. Jessica Keffer
Using Rhodamine Dimers to Explore the Role of Substrate Protein Binding on the GroEL Catalytic Cycle Despite being the most extensively studied
chaperonin protein, the effect of substrate protein (SP) binding to the GroEL catalytic cycle is not well understood because it contains the various levels of
heterogeneity: a given SP will have a variety of misfolded states, each of which can contain any number of GroEL-bound conformations. Our lab has
employed the use of tetramethylrhodamine (TMR) dimers as self-reporting molecular latches to address this problem. Using fluorescence or absorbance
measurements, the TMR dimers can be used to monitor the allosteric state of GroEL. Simultaneously, the dimers impose a mechanical constraint on the
enzyme, much like that of bound SP, that shifts the equilibrium between allosteric states, which stimulates ATPase activity. The uniformity of the non-covalent
intermolecular interaction within a dimer removes many of the levels of heterogeneity that accompany SP binding. This allows us to quantify the additional
energy the enzyme must exert in an allosteric state transition to overcome the mechanical constraint imposed by the TMR dimers.
University of Maryland, CONFORMATIONAL SAMPLING OF TELITHROMYCIN/CETHROMYCIN AND DESMETHYL ANALOGS Meagan C. Small1, Rodrigo B. Andrade2, and
School of Pharmacy
Alexander D. MacKerell, Jr.1 1 Department of Pharmaceutical Sciences, University of Maryland, Baltimore, MD 21201 2 Department of Chemistry, Temple
University, Philadelphia, PA 19122 Overcoming microbial resistance presents a major challenge in the development of new antibiotics. Approximately 50% of
all antibiotics target the peptidyl transfer center (PTC) of the bacterial ribosome, inhibiting protein synthesis and thereby disrupting cellular growth. A recent
class of 3rd generation macrolides, the ketolides, has been demonstrated to be effective against bacterial strains with macrolide- lincosamide-streptogramin B
(MLSB) resistance. This activity is due in part to the replacement of a C-3 cladinose sugar with a carbonyl, which bypasses a gene-encoded macrolide efflux
mechanism (mef) and addition of a alkyl-aryl chain to a C-11,12 cyclic carbamate, which increases ligand affinity via interaction with a second site. However,
ketolides are still susceptible to resistance via mutation mechanisms, the most common of which is A2098G (E.coli numbering). Crystallographic studies show
that this is due to the presence of a C-4 methyl group that sterically clashes with G2098. To probe the role of the C-4 methyl group as well as those at C-8 and
C-10 for structural simplification purposes, 20 nanosecond molecular dynamics simulations in explicit solvent were performed on telithromycin and
cethromycin and their desmethyl analogs. Analysis of selected distances distributions revealed that the conformations sampled by the parent compounds and
their desmethyl derivatives are significantly different. The probability maxima of the distances for both telithromycin and cethromycin overlap well with crystal
structure distances. Surprisingly, the largest difference is observed for the least chemically modified analog in both ketolides studied, C-4 desmethyl(ceth/teli)
thromycin. In addition, for b
Psoralen photochemistry illuminates cellular pathways of DNA interstrand crosslink repair. withdrawn
LMG, NIA, NIH
University of Maryland
UPenn School of
Medicine
Preferred deamination of C2’-endo cytidine by AID provides a mechanistic rationale for DNA deamination model Activation induced cytidine
deaminase (AID) deaminates cytosine to uracil, initiating the maturation of antibody responses through somatic hypermutation and class switch
recombination. Upon its initial discovery, AID was assumed to deaminate RNA, given its homology to APOBEC1, a known mRNA editing enzyme and its
robust ability to bind RNA. Subsequently, significant genetic and biochemical evidence has suggested that AID is exclusively a DNA cytosine deaminase. As
the lack of a mechanistic explanation for such discrimination has prompted ongoing debate, we have aimed to resolve the nature of nucleic acid selectivity. A
series of oligonucleotides were synthesized that contain a single embedded cytosine nucleobase linked to varied 2’-substituted furanose sugars. Using a
novel assay, tolerant to variations in the sugar, we have evaluated the ability for AID to deaminate cytosine presented in varied sugar pucker conformations.
Notably, AID demonstrates a marked selectivity for deamination of substrates with predominant sugar pucker conformations similar to DNA, while it is
inefficient at deaminating substrates with a favored RNA-like conformation. These data provide a structural rationale for the selective deamination of DNA,
despite the indiscriminate binding of either RNA and DNA by AID, and provide mechanistic support for the DNA-deamination model of antibody maturation.
University of Delaware Probing the Substrate Specificity of Quiescin-Sulfhydryl Oxidase Jennifer A. Codding and Colin Thorpe, Ph.D. Abstract The sulfhydryl oxidases support
the oxidative folding of proteins destined for secretion by oxidizing pairs of cysteine residues to disulfide bonds. Members of the Quiescin-sulfhydryl oxidase
(QSOX) enzyme family introduce disulfide bonds directly into protein substrates of higher eukaryotes. To gain a further understanding of the role of QSOX in
vivo, we here investigate the protein substrate specificity of QSOX in vitro. In particular, we have evaluated QSOX’s ability to form intramolecular and
intermolecular disulfide bonds with folded, unfolded, and partially folded proteins. We find that QSOX is unable to react with thiols buried in the interior of
proteins, but is highly active towards thiols in conformationally-flexible regions. The implications of these data for the physiological roles of the QSOX
enzymes, both within the secretory apparatus and outside the cell, are reviewed.
University of
Reducing the size of spectroscopic probes can increase the spatial resolution of fluorescence experiments on protein dynamics. We have shown pPennsylvania
cyanophenylalanine (Cnf) and backbone thioamides to be a fluorophore/quencher pair and we have determined their working distance to be 8-30 Å. We have
used this pair to study the thermal denaturation of a Cnf/thioamide-labeled version of villin headpiece by fluorescence spectroscopy. We are currently
exploring quenching interactions with other fluorophores and applying the method to monitor binding and folding in a few benchmark proteins. The small size
of the thioamide probe opens the possibility of mapping conformational changes with a density far beyond what is currently possible.
National Institutes of
The Chemistry and Biology of FtsZ Inhibitors Usable antibiotics for drug-resistant microbes are limited, and new antibacterial agents with novel
Health & Georgetown mechanisms of action and biological targets must be developed. An emerging target for antibacterials is the bacterial cell division protein FtsZ, the prokaryotic
University
structural homologue of tubulin. FtsZ is conserved among all pathogenic bacteria, and inhibition of its functions will shut down proliferation. FtsZ filaments
assemble to form the Z ring in bacteria, which is scaffold for the divisome complex. Inhibition of FtsZ has potential therapeutic use, but to date, no inhibitors
are clinically available. Our lab has recently isolated a novel class of antibacterial agents, termed the chrysophaentins, with potent antibacterial activity against
drug-resistant and drug-susceptible Gram positive organisms and no cytotoxicity towards eukaryotic cell lines. We used spectroscopic techniques to
determine the structures, and delineate the structure-activity relationship. Structural similarities with known FtsZ inhibitors led us to investigate the
chrysophaentin’s effects on FtsZ. In vitro enzymatic activity assays showed the chrysophaentins inhibit GTP hydrolysis in a dose-dependent manner with an
IC50 of 9.9 micromolar. Furthermore, transmission electron microscopy verified the disruption of GTP-dependent FtsZ filament formation in the presence of
the chrysophaentins. Saturation Transfer Difference NMR confirmed the chrysophaentins bind to FtsZ in a competitive manner with an analogue of GTP.
Finally, in a tubulin polymerization assay, the chrysophaentins had no effect, indicating the interaction with FtsZ is specific. These results are defining how the
chrysophaentins fit into the larger picture of FtsZ inhibitor modes of action.
55 Ms. Stephanie Schaefer
56 Ms. Beth Zucconi
57 Dr.
Georges Lahoud
58 Dr.
Reiko Sakaguchi
59 Ms. Gizem Keceli
60 Mr. Pavan Kumar
Mantravadi
University of Delaware Insertion of selenocysteine into the redox-active cysteine-x-x-cysteine motif of the flavoprotein augmenter of liver regeneration Stephanie A.
Schaefer, Renee Rubenstein, Colin Thorpe, Sharon Rozovsky Augmenter of liver regeneration (ALR) is a mitochondrial enzyme that aids disulfide bond
insertion into reduced unfolded proteins. ALR employs two active site cysteines to facilitate the formation of disulfide bonds. The reactive cysteines are part of
a cysteine-x-x- cysteine redox motif (where x stands for any amino acid) that is essential for activity. In order to study the catalytic mechanism of ALR we have
modified the redox potential of ALR by introducing a selenocysteine in place of a cysteine within the cysteine -x-x- cysteine motif. Selenocysteine is a naturally
occurring amino acid whose pKa and redox potential are lower than that of cysteine. Hence, while this mutation introduces no structural changes, the redox
potential of the modified ALR is expected to be substantially lower than that of the wild type protein. Selenocysteine was incorporated into ALR by
manipulating the Escherichia coli selenium incorporation machinery. As expected, the UV-VIS absorbance spectrum of the cysteine-x-x-selenocysteine
containing enzyme is shifted from 456 to 450 nm, indicating a different environment for the flavin cofactor. In contrast to wild type ALR, the selenium
containing enzyme forms a charge-transfer band in addition to a semiquinone complex when reacted with a small dithiol substrate. These spectral effects are
attributed to the interaction of the selenocysteine with the cofactor FAD while the enzyme was turning over.
University of Maryland, DIVERSE ASSOCIATION PARADIGMS OF THE AUF1 ISOFORMS INDUCE DIVERGENT mRNA STRUCTURES Beth Zucconi, Jeff Ballin, Brandy Brewer,
Baltimore
Christina Ross, Jun Huang, Eric Toth, Gerald Wilson AUF1 is a nucleic acid-binding protein that regulates the stability and translational potential of many
mRNAs. AUF1 binds to AU-rich elements (AREs) which are common in mRNAs encoding many proteins involved in cardiovascular, inflammatory, and
neoplastic diseases. There are four isoforms of AUF1 resulting from alternative splicing of exon 2 and/or 7 from a common pre-mRNA. Preliminary evidence
suggests that the different isoforms have varied functional characteristics, but no detailed, quantitative analysis of the properties of each has been reported
despite their differential expression. We used chemical crosslinking and gel filtration chromatography to show that each isoform exists as a high affinity
homodimer in solution. We then defined the association mechanisms of each isoform for an ARE-containing RNA substrate and quantified binding affinities
using fluorescence anisotropy and electrophoretic mobility shift assays which varied dependent upon the alternative exons. Finally, fluorescence resonance
energy transfer (FRET)-based assays showed that the different isoforms remodel bound RNA substrates into divergent structures as a function of
protein:RNA stoichiometry. We predict that alterations in the local structure of AUF1-substrate mRNAs will impact accessibility for ancillary trans-acting RNAbinding proteins and microRNAs proximal to AUF1 binding sites, which implies a potential role for AUF1-bound mRNA structures in priming posttranscriptional regulatory events. Together, these data describe isoform-specific characteristics among AUF1 ribonucleoprotein complexes, which likely
constitute a mechanistic basis for differential isoform function and regulation.
Thomas Jefferson
Post-surgical infections constitute a serious threat following all heart surgeries, with an estimate of 25-27% of infections occuring after surgeries. PostUniversity
operative infections are largely acquired in hospitals by opportunistic pathogens that are resistant to common antibiotics. Therefore the search for new
generations of antibitotics is in desperate urgency. Here we aim to delineate the differential separation of a pair of analogous proteins as a basis for selective
targeting of the bacterial species by a new generation of antibacterial agents. Bacterial TrmD and eukaryotic-archaeal Trm5 form an exemplary pair of
analogous proteins that introduce the m1G37 modification in tRNA to assure the fidelity of protein synthesis. These enzymes are structurally distinct, although
they use AdoMet as a common methyl donor to catalyze the same activity. Here we show that several naturally existing and synthetic analogs of AdoMet are
unable to adequately distinguish TrmD from Trm5. Instead, fragments of the methyl donor, adenosine and methionine, are significantly more inhibitory of
TrmD than Trm5. Structural information of the two enzymes in complex with adenosine reveals how Trm5 uses its dynamics to adopt global structural
changes, thus escaping targeting, whereas the rigid TrmD structure is enticed to accommodate the fragment and thus trapped by targeting. This work
demonstrates that chemical structures of substrate fragments can be exploited to identify analogs that functionally distinguish analogous enzymes to provide
a basis for structure-guided drug optimization programs.
Thomas Jefferson
Probing a tRNA Target Base for Methylation by Atomic Substitution The N1 methylation of guanosine 37 (m1G37) in tRNA is essential for maintaining
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the fidelity of translation and is required for cell growth in bacteria and in eukaryotes. The enzyme responsible for the synthesis of m1G37 is TrmD in bacteria
and Trm5 in eukaryotes and archaea. While TrmD and Trm5 catalyze the same reaction using S-adenosyl-methionine as the methyl donor, they have
different active-site structures. However, the molecular basis of how their distinct active sites recognize the target base is unknown. Understanding this
difference will offer an important foundation to develop novel antibiotics that selectively target TrmD. We have developed an approach to probe the recognition
of atomic groups in G37 by E. coli TrmD and M. jannaschii Trm5 as a model pair. In this approach, tRNA substrates with a specific atomic group substitution
were created by a combination of chemical synthesis and enzymatic reconstitution. The activity of each modified substrate was monitored by the singleturnover rate constant of methyl transfer (kchem) and by the kinetic equilibrium binding constant (Kd) of the enzyme-tRNA complex. The kinetic parameter
kchem/Kd of the normal over modified tRNA substrate is used to determine how TrmD and Trm5 discriminate the normal from the modified substrates. This
approach has given new insights into the molecular mechanism of substrate recognition by TrmD and Trm5.
Johns Hopkins
Reversibility of HNO-Induced Sulfinamide Formation Gizem Keceli† and John P. Toscano†,* †Department of Chemistry, Johns Hopkins University, 3400
University
North Charles Street, Baltimore, MD 21218 Cysteine residues are susceptible to several post-translational modifications under oxidative conditions. HNO, a
potential heart failure therapeutic, is known to be very thiophilic. The reaction of HNO with thiols results in the formation of disulfide or sulfinamide, depending
on the concentration of thiol. Traditionally, sulfinamide modification is considered to be irreversible in peptides and proteins. We have investigated the
reversibility of HNO-induced sulfinamide formation in small organic molecules, peptides and proteins under physiological conditions. Proteins are known to
undergo succinimide-mediated Asn deamidation at physiological pH and temperature. We hypothesized that the reversibility of sulfinamide modification might
be facilitated in peptides via a similar mechanism. It is shown that, in the presence of reducing agents, sulfinamide formation is reversible. Moreover, the
sulfinamide reduction is increased by at least a factor of three in the peptide structure vs. in small organic molecules. Although peptide sulfinamides are prone
to hydrolysis, it is slower than the reaction with thiols. Activity studies with a cysteine protease, papain, demonstrates the feasibility of sulfinamide reduction in
a protein environment. We conclude that there is a significant contribution from a low energy, cyclic intermediate (analogous to that in Asn deamidation) in the
reversibility of peptide sulfinamides.
University of Delaware Chemical acetylation of hormone receptors with biomimetic protein acetyltransferases Acetylation has increasingly been shown to be an important
post translational modification for proteins. Acetylation is involved in the regulation of a number of steroid hormone receptors. Acetylation of lysines on the
surface of steroid hormone receptors have been known to modulate hormone responsive behavior and is associated with a number of drug resistant cancers.
As natural protein acyltransferases function largely through proximity directed reactions, we have designed reactive ligand conjugates that when bound
position a thioesters in close proximity to reactive lysines on target proteins. Proximity directed acytlation represents a new paradigm in hormone receptor
modulation by small molecules.
61 Ms. Robin Samuel
62 Mr. Takwen Kajih
63 Ms. Sarah Grieshaber
64 Ms. Nandita Bhagwat
65 Ms. Winifred Johnson
66 Mr. Jun Liu
George Washington
University
Fluorescence recovery after photobleaching (FRAP) is a powerful microscopy technique to examine diffusion and flow processes in lipid
membranes. The experiment proceeds as follows: several pre-bleach scans are collected, a small area is bleached with a high intensity laser, and the
recovery of the fluorescence signal is monitored with respect to time. Fluorescence intensity over time corresponds to local exchange of fluorescent molecules
attributed to diffusion or directed flow. The extent to which diffusion changes after some perturbation to the lipid environment tells us a great deal about the
cross linking, non-specific binding, and or protein aggregation. We have applied this method to membranes containing biotinylated lipids that we cross linked
with avidin.
USAMRICD
Stereoselective Hydrolysis of G-Agents by a Chimeric Recombinant Paraoxonase 1 Enzyme Takwen T. Kajih, Sean M. Hodgins, J. Richard Smith#,
Vivekanand Shete*, Thomas J. Magliery*, David E. Lenz, Tony E. Reeves, Douglas M. Cerasoli and Shane A. Kasten Physiology and Immunology Branch,
Research Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5400 # Medical Diagnostic and
Chemical Branch, Analytical Toxicology Division *Department of Chemistry, Ohio State University ABSTRACT To generate a novel defense against poisoning
by organophosphorus (OP) nerve agents, a chimeric recombinant paraoxonase 1 (PON1) variant (MG-2-IA4) was generated by gene shuffling and multiple
rounds of directed evolution to be expressible in bacteria and to have enhanced catalytic efficiency for the hydrolysis of G-type OPs. We have assessed the
stereoselectivity of MG-2-IA4 for the more and less toxic isomers [P(-) and P(+), respectively] of the OPs GA, GB, GD and GF using a chiral gas
chromatography / mass spectrometry approach. For racemic GD and GF, MG-2-IA4 displayed a modest preference for hydrolyzing the more toxic P(-) isomer
(s). No stereopreference was detected with either GA or GB; both stereoisomers of each OP were hydrolyzed with almost equal relative catalytic efficiency. In
vivo efficacy studies conducted using guinea pigs administered different doses of MG-2-IA4 via a carotid catheter prior to subcutaneous exposure to 2 x
LD50s of GA, GB, GD, or GF showed that pretreatment with an adequate dose of this enzyme afforded protection without any signs or symptoms of
intoxication in exposed animals. At lower doses of enzyme, protection was observed against GA and GF but not GB or GD. Interestingly, the relative catalytic
efficiencies do not correlate precisely with in vivo protective efficacy. Additional work to define both in vitro and in vivo parameters for MG-2-IA4 is ongoing.
University of Delaware Elastin mimetic, cell adhesive multiblock hybrid copolymers Abstract: Biocompatible elastomers that not only capture the elasticity of native elastin but
also provide flexibility and tunability in a range of morphological, biological, and mechanical properties are needed for use as scaffolds for tissue repair and
regeneration. Hybrid polymer-peptide materials offer greater tunability in mechanical and other material properties from the presence of synthetic polymer
domains, while maintaining the structural and biological properties of the peptide domains. Elastin mimetic hybrid polymers (EMHPs) were synthesized via
condensation polymerization of azide-terminated poly(ethylene glycol) with alkyne-functionalized peptides by orthogonal click chemistry. The peptide was
composed of multiple alanine residues regularly spaced by lysine and terminally tagged with the RGDSP sequence derived from fibronectin in order to
promote cell attachment. The EMHPs were then crosslinked through the peptide lysine residues to form elastomeric hydrogels with mechanical properties
comparable to natural elastin. Neonatal human dermal fibroblasts (NHDFs) adhered to the EMHPs within one hour in serum-free media, and F-actin staining
showed the development and spreading of F-actin filaments after 24 hours of cell adhesion. Vinculin staining revealed that the cells attached to the gels via
focal adhesions. Cell proliferation was significantly greater on the RGDSP EMHP hydrogels in comparison with non-RGDSP EMHP hydrogels, and
competitive binding experiments showed that cell attachment was inhibited by incubation of NHDFs with soluble RGDSP peptide or RGDSP EMHP, indicating
the involvement of the RGDSP domains in integrin binding and cell attachment. These novel elastomeric, biocompatible EMHPs are promising candidates as
conducive scaffolds for vocal fold tissue engineering.
University of Delaware Electronic properties of peptide scaffolds containing PPV based chromophores as side chains Previous studies show that the alignment of the
chromophores and the distance between them are crucial parameters which govern the structure-property relationships in electronically active molecules
which are being studied extensively to enhance the performance of organic and polymeric semiconductor materials. However it is difficult to achieve control
for this purpose at angstrom length scales. In this work, we have employed helical peptides as scaffolds to organize oxadiazole-containing phenylenevinylene
(PPV) oligomers at desired spacing to study the interaction between these functional groups. The oxadiazole-containing PPV oligomers will be coupled at
specific positions on alanine-rich helical peptides. This system is designed to incorporate up to three chromophores strategically placed on the peptide
backbone in the range of 6-17Å from each other. Circular dichroism spectroscopy is used to confirm the helical nature of the peptide. Other spectroscopic
techniques such as exciton-coupled CD, photoluminescence, and absorption will elucidate how the chromophores interact with each other when placed on the
same side of the peptide chain or on opposite side of the peptide chain.
Porphyrin-Peptide Self-Assembling Nanowires We have designed and synthesized two porphyrin-peptide systems that self-assemble. Our goal is to
Haverford College
create a nanowire that efficiently transfers energy using exciton coupling in a manner analogous to energy transfer between chlorophylls in the photosynthetic
antenna complex of bacteria and plants. We have synthesized a 10-mer peptide, NAEASAESAY, and coupled two monoaminotetraphenylporphyrins to the
glutamic acid residues. This system shows ordered aggregation and increased !-helical character as observed with UV-Vis Spectroscopy, CD Spectroscopy,
and Atomic Force Microscopy (AFM). We are also working with a system that couples a 7-mer peptide, GNNQQNY, with Deuteroporphyrin IX 2,4-bis ethylene
glycol at the N-terminus of the peptide. The 7-mer peptide is known to form "-sheets and based on observation and UV-Vis we see extensive aggregation. At
pH 4 we observe a characteristic "-sheet peak in IR as well as potential fibril formation according to AFM studies. We are now studying a new peptide,
GSFSIQYTYHV, which is known to form a hydrogel. AFM indicates that this peptide forms large fibrils which seem promising as a structural framework for
porphyrin. We are also exploring a new synthetic approach in this system where the porphyrin is coupled to the peptide before cleavage from the resin.
University of Delaware Structure and Function of Selenoprotein K Jun Liu, Prabhavathi Srinivasan, and Sharon Rozovsky University of Delaware, Department of Chemistry and
Biochemistry, Newark DE 19716 Selenoprotein K (SelK) is a membrane selenoprotein that is critical to longevity. Selenoproteins contain the genetically
encoded amino acid selenocysteine that is predominantly found in enzyme active sites where it provides high chemical reactivity and specificity. The function
of SelK is unknown but it was shown to participate in anti-oxidant defense in vivo, possibly in the Endoplasmic Reticulum Associated Protein Degradation
(ERAD) pathway. To facilitate SelK structural and biophysical characterization, we have developed bacterial overexpression strategy using a fusion partner to
stabilize and solubilize SelK. The fusion protein can be cleaved from SelK in the presence of a variety of different detergents compatible with structural
characterization by nuclear magnetic resonance spectroscopy. Furthermore, we demonstrate the incorporation of selenium in SelK forming the active
selenoprotein.
67 Prof. Karin Akerfeldt
68 Dr.
Haijuan Du
69 Dr.
Pumtiwitt McCarthy
70 Mr. Yang Liu
71 Mr. Sean Hodgins
72 Ms. Nwe-Nwe Aye-Han
73 Mrs. Cay Tressler
Haverford College
Characterization of a Semenogelin I Peptide Hydrogel Jamie DeNizio(1), Birgitta Frohm(2), Sara Linse(2) and Karin Åkerfeldt(1) (1) Department of
Chemistry, Haverford College, Haverford, PA 19041 (2) Department of Biochemistry, University of Lund, Sweden The aim of this project is to investigate the
hydrogel forming properties of a sequence derived from Semenogelin I, a protein found in seminal plasma. Hydrogel formation tests were performed varying
the peptide concentration, solvent, pH , salt concentration, Zn2+ concentration, and temperature. Circular dichroism (CD) and FT-IR spectroscopy were used
to determine the secondary structure, and atomic force microscopy (AFM) was used to look at the tertiary morphologies of the peptides in solution. It was
found that the 11 residue sequence forms a hydrogel dissolved in water slightly above neutral pH. The CD spectra confirm the hypothesis that the peptide
self-assembles to form b-sheets, both initially at acidic pH and after gel formation at basic pH. In addition, the IR spectra indicate strong b-sheet
characteristics at basic pH, exhibiting extended sheets after the sample had gelled. The AFM images display different morphologies depending on substrate,
incubation time and temperature. Current work includes the synthesis and purification of related sequences with an eye to increase peptide cross-linking in
order to form more durable hydrogels.
George Washington
Insights of the intrinsically unstructured C-terminal domain of alpha4, regulator of MID1 and PP2A The structural properties of the C-terminal residues
University
(Glu236-Leu280, alpha45) of Alpha4 are probed by NMR spectroscopy. We observed that even though there were no strong NH-NH(i,i+1) NOEs indicative of
helical elements, chemical shift index suggested the presence of one alpha-helix located on the C-terminus. Analysis with TALOS+ confirmed this
observation. Interestingly, we observed that alpha45 adopted two alpha-helices in the solution with 1 % SDS. The residues that formed the alpha-helices were
also predicted to be helical and identified by CSI and TALOS+ to be helical. HSQC experiment revealed that alpha45 binds tightly to the Bbox1 domain from
MID1 in aqueous solution. Together, these results suggest that the C-terminus has intrinsically unstructured properties.
CBER, U.S. Food and Preparation of a meningococcal group C polysialic acid- tetanus Hc fragment glycoconjugate vaccine candidate by chemoenzymatic synthesis Global
Drug Administration
immunization efforts against meningitis have been effective at reducing incidence of the disease worldwide. Early meningitis vaccines, composed of acidic
capsular polysaccharides of disease-causing bacteria, elicited a strong, long-lasting antibody response in adults but not in children. Current strategy uses
these capsular polysaccharides attached to a carrier protein to increase response in children. However, the chemical methods used to make these vaccines
lead to heterogeneous mixtures. Well defined vaccines are needed to elucidate the relationship between vaccine structure and generated immune response.
Here we describe our efforts to produce these types of structures using chemoenzymatic synthesis. Chemically synthesized lactosides, containing azido
groups at the aglycon were used as substrates for recombinant sialyltransferase enzymes from Camplyobacte
University of Maryland Selective alkylation of DNA by PNA-QMP Quinone methides (QMs) can react with a variety of nucleophiles including those in DNA under physiological
conditions. In order to create sequence specific alkylation, a PNA-QMP (peptide nucleic acid - quinone methide precursor) has been generated. PNA is a
peptide-based analogue of DNA which can bind to complementary DNA by Watson-Crick and Hoogsten base pairing and can be used as a delivery agent for
target-directed alkylation. Such alkylation of ssDNA has now been detected by denaturing polyacrylamide gel electrophoresis (PAGE). PNA-QMP
demonstrates significantly more reactive with a fully complementary DNA than with a sequence containing a single base-mismatch. A reversible PNA-QM
self-adduct has also been generated and purified by HPLC.
University of Baltimore In Vivo and In Vitro Binding of Organophosphorus Nerve Agents by Human Butyrylcholinesterase (BuChE): Role of Stereoselectivity in Protective
Efficacy Sean M. Hodgins, Shane A. Kasten, Zeke P. Oliver, Monika A. Kauffman, Takwen T. Kajih, Tony E. Reeves, David E. Lenz and Douglas M. Cerasoli
County/Medical
Physiology and Immunology Branch, Research Division, U.S. Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, APG, MD
Research Institute of
21010-5400 Current treatments for organophosphorus (OP) nerve agent exposure act by blocking synaptic receptors of the neurotransmitter acetylcholine
Chemical Defense
(ACh), reactivating inhibited acetylcholinesterase (AChE), and halting or preventing seizures. While effective in protecting against lethality, these interventions
result in behavioral and performance impairments. An alternative approach that targets and removes OPs from the circulatory system prior to AChE inhibition
would provide advantages over conventional therapy. Human butyrylcholinesterase (BuChE), which is structurally and functionally related to AChE, has been
shown to effectively scavenge nerve agents in circulation and provide asymptomatic protection against otherwise lethal exposures to OPs. OP nerve agents
all have a stereocenter at the phosphorus atom, and the Ps isomer of each is more inhibitory of AChE in vitro and more toxic in vivo. Using chiral liquid and
gas chromatography coupled with mass spectrometry, it was found that for some OPs, BuChE and AChE display similar stereoselectivity for the Ps isomers.
We also determined the extent of protection against OP toxicity (protective ratio; PR) afforded by administering BuChE to male Hartley guinea pigs via
intramuscular or intravascular injection. Human BuChE from two different sources (outdated human blood or the milk of transgenic goats) was tested, and the
PR at different times after enzyme injection was directly proportional to the amount of BuChE activity in circulation at the time of OP exposure. The PR
afforded by BuChE against those OPs for whic
Johns Hopkins
Protein kinases are key nodal signaling molecules that regulate a wide range of cellular functions. Most of the time, these enzymes are regarded as simple
University
on-off switches that control linearly structured signaling pathways. Yet, there have been various reports of differential substrate phosphorylation by the same
kinase in a context dependent manner. Such diverse roles of protein kinases require spatial compartmentalization as well as temporal regulation of their
activity. Here, using FRET-based activity reporters and intracellular Ca2+ dye, we have shown that protein kinase A (PKA), Ca2+ and cAMP oscillate in sync
and form a highly integrated oscillatory circuit in insulin secreting MIN6 "-cells. We found that PKA was capable not only of initiating the oscillatory circuit but
also modulating its frequency, thereby diversifying the spatiotemporal control of its downstream signaling. Using various inhibitors of PKA signaling pathways,
we are dissecting the underlying mechanisms that lead to changes in oscillatory behaviors of cAMP, PKA and Ca2+. Our findings suggest that exquisite
temporal control of kinase activity, mediated via signaling circuits resulting from cross-regulation of signaling pathways, can encode diverse inputs as temporal
parameters such as oscillation frequency, which in turn can ensure proper regulation of complex cellular functions in a context-dependent manner.
University of Delaware Design of Minimal Protein Kinase-Inducible Domains for Detection of Kinase Activity Cellular pathways often require post-translational modifications,
such as phosphorylation, to regulate cell signaling. Errors in cell signaling often result in diseases such as cancers and Alzheimer's disease. Part of
understanding these pathologies is developing better probes to examine kinase activity. Ideally, these kinase probes should be genetically encodable as well
as sensitive. It has been shown that phosphorylated peptides can easily be differentiated from their unphosphorylated counter-parts by terbium fluorescence
by coordination in the EF-hand motif. The EF-hand coordinates metal at positions 1, 3, 5, 7, 9, and 12. A key design element of these sensors is the
phosphoserine at position 12. Most of these sensors are longer than fifteen amino acids in length. The movement of the phosphorylation site to position 9
shows an increase in terbium binding, especially in the instance of phosphoserine. Fluorescence differentiation was even further increased by the substitution
of lysine at position 2. Multiple minimal protein kinase-inducible domains were developed using combinations of these design elements and optimized as
phosphorylation sensors for multiple targets.
74 Mr. Christopher McGann Materials Science
Modular Resilin-like Polypeptides as Tissue Engineering Scaffold for Mechanically Active Tissues Christopher L McGann1, Linqing Li 1 and Kristi L.
University of Delaware Kiick1,2 1Department of Materials Science and Engineering University of Delaware, Newark, DE, 19716, USA 2Delaware Biotechnology Institute, 15
Innovation Way, Newark, DE 19711 Abstract: Resilin is an important elastomeric structural protein found in insects that facilitates the function of mechanically
active tissues; specifically, the protein plays a role in insect flight, sound production, locomotion and energy storage. Resilin contains a motif sequence that
has been previously demonstrated to provide highly elastic and resilient properties when cross-linked into hydrogels. Previously, we have demonstrated the
design and expression of a recombinant modular resilin-like polypeptide (RLP) with the both the elastic structural domains as well as additional sequences
conferring MMP degradation, integrin recognition/binding and heparin/VEGF binding properties. In this work, higher molecular weight RLPs were created by
multimerizing the original gene through recombinant DNA techniques in order to investigate the mechanical properties of higher molecular weight RLPs.
Additionally, these high molecular weight RLPs enhanced the ability to investigate the sequences encoding for biological properties such as the MMP
degradation and the heparin-binding domain.
75 Mr. Eric Levenson
University of Delaware Probing the molecular mechanism of Toll-like receptor 9 activation Toll-like receptor 9 is an innate sensor of extracellular surced DNA from bacteria,
fungi, and ds viruses. The two main classes of ssDNA used to activate TLR9, known as CpG-A and CpG-B, differ based on their ability to form larger
aggregates (CpG-A) or to remain monodisperse (CpG-B). CpG-A causes strong interferon production from within LAMP2+ endosomes while CpG-B induces
cytokine production with cell maturation from within VAMP3+ staining endosomes, producing the antiviral and humoral or cell mediated responses
respectively. The mechanism for this localization and difference in cytokine response is unknown, but its elucidation would provide important targets for
adjuvants and immune therapies. We report the synthesis of defined nucleic acid and polymer conjugateswhich control the valency and density of nucleic acid
as well as polymer length to delineate the mechanisms responsible for the differential cytokine release in pDCs and macrophages. These polymers are
produced via a combination of controlled polymer synthetic methods and bioconjugation techniques. We report the ability of these conjugates to enhance
immunogenicity of the Toll-like receptor 9 ligand CpG DNA 232Ms.Katherine BeleckiJohns Hopkins [email protected]
76 Mr. Qing Liu
Highly fluorescent porous silica nanoparticles were synthesized in a water/oil phase using organic templates method. In this work, we introduce a one-step
method to label porous silica nanoparticles (PSN), with long term stability, using polystyrene (PS) as the pore template, cetyltrimethylammoniumbromide
(CTAB) as a micelle template, and an embedding “lipid” dye(Liss Rhod PE: dye LR, and Dansyl PE: dye D). The use of a lipid like dye enables it to function as
a surrogate surfactant with the primary surfactant CTAB on the organic and inorganic interface. Also, this approach allows us to independently control both
pore and particle size by adjusting template mononer styrene amount and water/oil ratio. All above make it an important work in biological applications.
234Mr.Aaron LeeUniversity of [email protected]
77 Ms. Jennifer Adler
Univ. of Maryland
Iodide salvage is essential to thyroid hormone metabolism and metabolic regulation. In mammals, the DEHAL1 gene product iodotyrosine deiodinase
(IYD) is responsible for deiodination of the mono- and diiodotyrosine byproducts of thyroid hormone synthesis (triiodothyronine and thyroxine, T3 and T4). IYD
is a membrane-bound flavoprotein comprised of three domains with the catalytic domain belonging to the NADH oxidase/flavin reductase structural
superfamily. This enzyme has been expressed in mammalian, insect, yeast, and bacterial cell lines. Mammalian and insect cell lines were used to express the
native gene sequence while yeast required codon optimization, and bacteria required additional engineering. CD spectra, kinetic rate constants, binding
constants of substrates, and a series of crystal structures were obtained of IYD. Analysis of the crystal structure of IYD indicates a dimer with an active site
comprising of both monomers orienting the C-I bond of iodotyrosine substrate stacking above the N5 of the flavin mononucleotide (FMN) required for activity.
Three amino acids (E153, Y157, and K178) in the active site form hydrogen bonds and electrostatic contacts with the zwitterionic portion of the substrate.
Mutation to any of these three amino acids significantly decreases substrate-binding affinity and enzymatic activity.
78 Mr. Chris Weinacht
University of Delaware Dynamic Orientation of Ubiquitylated PCNA In Solution and Its Implication in Translesion Synthesis Chris Weinacht1, Junjun Chen1, Yongxing Ai1,
Susan E. Tsutakawa2, Ivaylo Ivaynov3, John A. Tainer2, Zhihao Zhuang1 1. Department of Chemistry and Biochemistry, University of Delaware, Newark,
DE, 19716; 2. Life Science Division, Lawrence Berkeley National Laboratory, Berkeley, California, 94270; 3. Department of Chemistry, Georgia State
University, Atlanta, Georgia 3030 Translesion synthesis (TLS), a mechanism utilized by cells to synthesize past DNA lesion, is evolutionarily conserved in
organisms from prokaryotes to eukaryotes. Ubiquitylation of proliferating cell nuclear antigen (PCNA) plays an important role in eukaryotic TLS. The molecular
details of how ubiquitylation of PCNA regulates TLS are not fully understood. One obstacle in studying eukaryotic TLS resides in the difficulty of preparing
sufficient amount of ubiquitylated PCNA. We developed a chemical approach for ubiquitylation of PCNA. The chemically ubiquitylated PCNA is functionally
equivalent to the native ubiquitylated PCNA in effecting polymerase switch. We also demonstrated the strict requirement of PCNA ubiquitylation for
polymerase exchange. Moreover, our study revealed a surprising degree of mobility of ubiquitin moiety on PCNA. To further determine the structure of
ubiquitylated PCNA in solution, we combined small angle X-ray scattering (SAXS) and multi-scale computational modeling. We identified three positions for
ubiquitin along the axis of the PCNA homotrimer. We argue that these flexible conformations are important for exposing the hydrophobic surface of ubiquitin
needed to bind the TLS polymerase. However, despite this high degree of flexibility, there are preferred positions on the PCNA ring with which ubiquitin
interacts. We find that the ubiquitin occupies a preferred position on the back face of the PCNA ring as observed in the X-ray crystal structure. We also find
the ubiquitin occupies a position on the side of the PCNA ring as suggested by the simulation. Finally a significant portion of the population is in a state where
the ubiquitin moieties are in a flexible position in which the ubiquitin is attached to Lys-164 of PCNA but is not otherwise interacting with the PCNA. An
understanding of this flexibility will be critical for understanding the recruitment and switch of TLS polymerase Pol &. This work was supported by a grant to
Z.Z. from National Science Foundation (MCB-0953764).
79 Mrs. Christina Ford
University of Delaware Synthesis of peptides containing 4-thiophenylalanine Although Nature has evolved a plethora of protein functions from only 20 common amino acids,
incorporation of non-natural amino acids into proteins can provide tools for reporting and tuning protein structure and function. We describe a coppercatalyzed reaction, which is compatible with all of the natural amino acids, that generates 4-thiophenylalanine on solid-phase without side products. By itself,
4-thiophenylalanine can function as a simple structural “switch” in proteins, where aromatic '–interactions can be strengthened or weakened an adjustment in
pH. One can also use simple reactions, such as those typically used in cysteine modifications, to generate a wide variety of aryl-thiol derivatives (including
thioethers, disulfides, sulfoxides and sulfones) and introduce greater functionalization on an aromatic amino acid. These derivatives can also be used to tune
local structure within a peptide, and we report these electronic effects with a number of novel derivatives in the context of the aromatic-prolyl interaction, which
is known to influence the Ktrans/cis of the amide bond by substituted aromatic residues adjacent to proline.