Undergraduate Research Symposium 2007: Abstracts
Step-wise Approach to Crystallization of Protein Kinase A Holoenzyme with RII Regulation Subunit
Kimberly A. Alberto, Simon H. J. Brown and Susan S. Taylor Department of Chemistry and Biochemistry, Howard Hughes Medical Institute,University of California, San Diego, La Jolla, Ca, 92093Phosphorylation is a classic signaling mechanism in biochemistry. Protein kinases allow eukaryotic cells to receive extracellular signals and translate those signals into biological responses by the use of phosphorylation. The activity of protein kinases is turned on and off by uniquely regulated inhibitory regulatory subunits that are the primary receptors of cAMP. Protein Kinase A (PKA) has two classes of regulatory subunits (RI and RII), with each class having two isoforms (. and .). RII is a substrate of the catalytic subunit (C) of PKA, while RI is a pseudosubstrate. Both the RI and the RII subunits have similar domain organization. At the C-terminus are two cAMP tandem binding domains. At the N-terminus is a dimerization/docking domain connected to an inhibitor site by a flexible linker region. It is in this linker region that diversity is seen between RI and RII. The inhibitor site for RII. is autophosphorlyated at Ser108 by the C subunit. The RII. (102-265) includes 10 residues N-terminal to the PKA inhibition site. This region was included to study the binding of this linker region of RII. and how it differs from RI. Here, the purification and crystallization of the holoenzyme of RII. (102-265) subunit is described. The RII. (102-265) subunit was expressed in E. coli. It was then purified by two methods of chromatography: affinity chromatography and fast protein liquid chromatography (FPLC). The purified protein was then screened in various crystallization conditions. The crystallization screen PACT produced at least twenty of the best conditions of all tested screens. The conditions with the best crystals were then optimized by varying the precipitant concentration, protein concentration, salt concentration, pH and by the technique of microseeding. Crystals from the optimization diffracted to 2.9Åand the structure of RII. (102-265) holoenzyme was solved.
Controlled Drug Delivery using Porous Silicon and Polycaprolactone Composite Materials
Christina I. Brady, Emily J. Anglin, Michael J. Sailor Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093Porous silicon- polycaprolactone polymer (PSi-PCL) composites are investigated as an implantable drug delivery material. Single-crystalline p-type silicon wafers (boron-doped) with varying resistivities were electrochemically etched to produce different pore sizes. The PSi-PCL composites were loaded with fluorescein as a model therapeutic agent, and in vitro dissolution studies were conducted. The PSi-PCL composite materials are flexible and provide uni-directional release of the drug surrogate dye. UV-visible spectrophotometry was used to measure the absorbance of fluorescein released into the surrounding solution. The composite material comprised of porous silicon made from Si with a resistivity of ~2 .·cm possesses smaller average pore diameters and a higher surface area that degrades faster than porous silicon prepared from lower-resistivity (~1 m.·cm) Si. This latter material has pores with a larger average diameter and has a smaller total surface area. Control films consisting of porous Si without polycaprolactone demonstrate an instantaneous release of the dye whereas a control consisting of a polycaprolactone polymer film displays a significantly slower dye release rate. The nanocomposite has intermediate release kinetics, which are different from either the PSi film or the PCL film alone.
The Role of Bordetella Reverse Transcriptase in Diversity Generation
Helen Chen and Partho Ghosh Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr. La Jolla, CA 92093Bordetella bacteriophage can generate a massive diversity in its retroelement-encoded receptor-binding protein, major tropism determinant (Mtd). The phage is capable of producing ~1013 possible sequences of Mtd, which enables the phage to adapt to changes as the host Bordetella transits through its infectious cycles. Previous works has shown that this massive diversity is a result of a cassette-based mechanism that is a template-dependent and reverse transcriptase-mediated process. It has also been found that the diversity is caused by a series of nucleotide substitutions at template adenines, and it is unclear whether the reverse transcriptase activity is sufficient for this adenine-directed mutagenesis. In this project, we examine the specific functions of the Bordetella reverse transcriptase on mediating diversity. The reverse transcriptase was expressed recombinantly in E. coli and purified from inclusion bodies. After refolding, its function was tested in vitro by an RT-PCR assay. Analysis on the reverse transcription products is ongoing and will give insight into the role of Bordetella reverse transcriptase in the diversity-generating mechanism.
Ultrasound-Induced Release of Dexamethasone from Hydrosilylated Porous Silicon Particles
Luis Ching, Emily J. Anglin, Michael J. Sailor Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr. La Jolla, CA 92093Porous silicon microparticles are investigated as an ultrasonically-induced drug delivery system. The particles are prepared by an electrochemical etch of single-crystalline P-type silicon wafers and subsequently modified by a thermal hydrosilylation reaction with 1-dodecene. The degree of surface modification is confirmed by Fourier transform infrared spectroscopy. Drug release from the particulate system is triggered by a short (3 min.) application of ultrasound and is monitored by UV-visible absorption spectrophotometry. Preliminary results demonstrate that the particles that were exposed to ultrasound showed a significantly greater release of drug (by a factor of 4) in solution as opposed to the release rates of the porous silicon particles that were not exposed to ultrasound.
Understanding a Cell Fate Decision in Terms of Homeostatic and Dynamic Control of Information Processing Signaling Pathways
Rebecca Delker, Paul Loriaux, Shannon Werner and Alexander Hoffmann Signaling Systems Laboratory, Department of Chemistry and Biochemistry, University of California at San DiegoCell fate in response to inflammatory stimuli is described as a balance of pro- and anti-apoptotic signaling mediated by the Caspase 8, JNK and NF-.B signaling pathways, respectively. These pathways are multiply interconnected through the function of the mediators, A20, ITCH, cFLIP, and redox control proteins. Depending on the strength of these crosstalk mechanisms, the same inflammatory stimulus can result in cell activation as well as survival or death. In order to gain a quantitative understanding of signal processing within this network and construct a mathematical model, we have measured dose response curves, and . using knockouts and specific pharmacologic inhibitors . quantitatively determined the role of signaling crosstalk between components. Our preliminary results indicate that cumulative JNK activity, including steady-state activity, determines the sensitivity to cell death. Due to a very steep dose response curve of JNK, even low constitutive NF-.B activity is sufficient to counteract the propensity to enter the death pathway by ensuring low levels of the negative feedback regulator A20 and redox control proteins. Similarly, homeostatic levels of FLIP appear to be more important than its stimulus-induced expression. However, cellular homeostasis depends on the cumulative history of exposure to stimuli. As such we are exploring the effect of priming cells with non-apoptotic stimuli on the decision to undergo cell suicide.
Preparation of Nano-porous Si Particles for In-vivo Imaging and Drug Delivery
Kim Duong, Mike Sailor Research Group Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093Porous silicon particles were formed by electrochemical etch of a p-type (boron doped) silicon wafer in an ethanolic hydrofluoric acid solution. We are interested in studying these particles for diagnosis and treatment of ovarian cancer. Nano-porous luminescent silicon particles were formed by etching at a current density of 124 mA/cm2 for 4 minutes followed by an electro-polishing step that removes the porous film from the silicon substrate. The porous film is broken up into micron-size particles by ultrasonication. The next step in my procedure is to chemically modify the porous particles such that drug molecules or a molecular imaging agent can be covalently attached to the inner pore walls of the particles. This is accomplished by hydrosilylation with an alkylcarboxylate. Conjugation of a dye to the carboxy terminus results in luminescent porous silicon particles. One of the important applications of nano-porous luminescent silicon particles in cancer research is in-vivo imaging. In addition, we hope to attach anti-cancer drugs to the particles so that they can be tested as therapeutics in our ongoing ovarian cancer studies.
Three-Legged Piano Stool Cp*M(CO)2(NO) Complexes, M = Mo and W
Jordan Fine and Torri Catalano Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093A series of three-legged piano-stool complexes, Cp*M(CO)2NO, M = Mo and W have been prepared and structurally characterized in which various large organic substituents on tetramethyl(organyl)cyclopentadienyl ring have been used to study the structural relationships of the organyl substituent and the nitrosyl leg. In most cases no preferential orientation was observed. Instead, most structures exhibited considerable, yet resolvable, CO/NO disorder.
The Diastereoselectivity of the Ugi Reaction in the Synthesis of Pyroglutamic Acid Derivatives
Breena Fraga, Cynthia B. Gilley, and Yoshihisa Kobayashi Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093Pyroglutamic acids are often found in the structure of natural products and also important precursors of total synthesis and asymmetric reaction. The Ugi 4-center 3-component condensation reaction of levulinic acid (CH3COCH2CH2CO2H) is known to yield this heterocyclic compound as a carboxamide. The conversion of the resulting C-terminal amides to the corresponding acids was not known in the literature due to the difficulty in hydrolyzing such a sterically hindered amide. Our laboratory recently introduced a novel convertible isonitrile which when employed in the Ugi reaction with .-ketoacids can facilitate the hydrolysis of the resultant pyroglutamic acid amide via an N-acylindole intermediate.1,2 This research investigates on the stereoselectivity in the multi component reaction of chiral .-hydroxy-.-ketoacids. The hydroxyl group on the stereocenter at the . position of the carbonyl component could be an effective directing group, thus inducing stereoselectivity in the Ugi reaction.
Spectroscopic Studies of Antimicrobial Peptides: Probing Peptide-Membrane Interactions
Jonathan Gable, Diana Schlamadinger, Katheryn Sanchez, Judy E. Kim Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA, 92093Antimicrobial peptides are a major component of the innate human immune system. Human neutrophil protein .-defnsin 1 (HNP-1), one of numerous human antimicrobial peptides, is ideal for spectroscopic study because of a single native tryptophan residue present in the 30-residue chain. Current research indicates that HNP-1 selectively disrupts bacterial cell membranes. However, a general mechanism of action has not yet been determined. We have used a combination of molecular biology and spectroscopic tools to study HNP-1 and its interaction with synthetic phospholipid bilayers. Specifically, we have developed an expression system to express mg-quantities of HNP-1, and are currently using steady-state and time-resolved emission techniques to probe interactions between HNP-1 and liposomes. Fluorescence leakage assays reveal membrane disruption upon binding of HNP-1 and anisotropy experiments indicate hindered mobility upon membrane association. Additional ongoing experiments include lifetime measurements as well as Raman spectroscopy. Collectively, these tools will allow for a better understanding of HNP-1 antimicrobial action on a molecular level.
Synthesis and Paramagnetic NMR Studies of Inorganic Mixed Valence Complexes: Electron Exchange Kinetics
James C. Hauk, Christina J. Hanson, and Clifford P. Kubiak Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093We are involved in the synthesis of tri-ruthenium clusters to study intermolecular self-exchange electron transfer rates. Practice studies were performed with the ferrocene-ferrocenium redox pair. Using 1D NMR and IR spectroscopy techniques we have characterized several tri-ruthenium clusters which contain a number of different ancillary ligands. Synthesis of mixed valence dimers of tri-ruthenium complexes has already been performed for the purpose of studying intramolecular electron transfer. Work done on said dimers found electron transfer rates on the ultra-fast (picosecond) time scale. By completing this project we will gain a more complete understanding of the reorganization energies for electron transfer between separate molecules and this will allow us to further understand the observed properties of the ultra-fast ground state electron transfer exhibited by the dimers of these clusters.
A Ribonucleic Acid Nanosquare
Jennifer L. Johnson, Tracey Pham, Jerod Parsons & Thomas Hermann Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093We have designed a square-shaped RNA nanostructure of 100 nucleotides that self-assembles by multimerization of two short oligoribonucleotides. The architecture of the RNA nanosquare is based on four identical .nanocorners. which represent sharply bent double-stranded motifs that we discovered by X-ray structure analysis of a viral regulatory RNA element. The initial characterization of the RNA nanosquare by native polyacrylamide gel electrophoresis is presented as well as investigations of the metal ion-dependent self-assembly and crystallization trials towards structure determination by X-ray diffraction.
Design and Synthesis of a Fluorescent Peptidyl Aldehyde-Based Inhibitors of the Eukaryotic Proteasome
Jungman, R., Foley, T. and Michael D. Burkart Department of Chemistry and Biochemistry; University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093A fluorescently labeled peptide was synthesized using the Fmoc protecting ring on a solid phase polystyrene resin. A fluorescent coumarin molecule was attached to the peptide with varying length alkyl chains to monitor effectiveness in binding to the eukaryotic proteasome and inhibiting protein degradation. The terminal amino acid is reduced to an aldehyde, as geminal diols have shown effectiveness in mimicking protease substrate intermediates and binding to the active site of the proteasome.
Polythiophene Linked Single Molecule Magnets and Heat Capacity Studies of Ni4 SMMs
James M. Ma, Chris C. Beedle, David N. Hendrickson Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093Polythiophene is utilized as a link to join single molecule magnets (SMM) into magnetic chains. A nickel cubane SMM has been synthesized with pendant 2-thiophenemethanol ligands that will serve as the points for polymerization. A galvanostatic electrochemical setup has successfully polymerized thiophene and 2-methanolthiophene in an acetonitrile solution with TBAPF6 as the supporting electrolyte. Efforts are currently underway to oxidatively electropolymerize the SMM under similar conditions. Heat capacity studies are being used to determine the cause of a large rise in the magnetic susceptibility of the [Ni(hydroxymethylpyridine)(dimethylbutanol)Cl]4 SMM at 0.29 K.
Novel Tools for Studying Protein Turnover and Function
Michael McKeown, Michael Lin, Roger Tsien Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093Genetically encoded labeling of proteins to enable visualization of their location has become an important method in molecular biology. However, improving tools to report protein age and function would be useful to facilitate the study of more complex pathways. TimeSTAMP was developed to allow drug mediated selective retention of an epitope tag to a protein of interest. The tag is constitutively removed by a cis-acting protease until an inhibitor is applied, causing all newly synthesized proteins to remain tagged. Applying TimeSTAMP to study PSD-95 turnover in transfected neurons with microscopy and Western Blot analysis, we found that cell cultures stimulated by co-culturing with new neurons have an increased rate of PSD-95 synthesis. In separate work, we have been developing FRET reporters for multiple simultaneous signaling events. We have optimized green fluorescent proteins (GFPs) for improved spectral properties. A modified sapphire, which excites in the UV range, and a modified GFP called iGFP, which excites in the blue range, could be independently activated and used to transfer energy to a red fluorescent protein, enabling two FRET sensors in the same cell.
Purification and Functional Analysis of WISP3
Dustyn Miller and Malini Sen Department of Chemistry and Biochemistry, Department of Medicine, University of California, San Diego, La Jolla, CA, 92093WISP3 (Wnt induced secreted protein 3)/CCN6 is a member of the CCN (CTGF, CYR61, and NOV) family of connective tissue growth factors. Several mutations and splice variants of WISP3 have been linked to connective tissue disorders and various malignancies. Functional studies have suggested that WISP3 contributes to tissue maintenance and homeostasis. However, the precise molecular mechanism of WISP3 function remains unresolved. The present study was conducted to investigate the potential impact of WISP3 on the accumulation of reactive oxygen species (ROS) and oxidative stress, which are central to cell/tissue maintenance. We have expressed WISP3 in baculovirus and partially purified it. The recombinant WISP3 regulates accumulation of ROS.
Triggered Drug Release Using Composites of Porous Silica and poly(N-isopropylacrylamide-co-acrylic acid) Hydrogel
Troy Moore, Ester Segal, Loren A. Perelman, Michael J. Sailor Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr. La Jolla, CA 92093A pH and thermoresponsive poly(N-isopropylacrylamide-co-acrylic acid) hydrogel is polymerized within a nanoporous silica template. Scanning electron microscopy and spectral analysis of the composite show that the hydrogel is integrated into the nanostructure while retaining its characteristic volume phase transition, at which a reversible collapse/expansion of the hydrogel occurs. These composites are studied as potential drug delivery platforms. The phase transition of the composite is examined with respect to the loading of bovine serum albumin or bovine pancreatic insulin into the composite, followed by subsequent release of the protein. The loading of each occurs via passive diffusion into the hydrogel after submerging the composite in solutions containing dissolved quantities of each protein. The release of the protein from the composite is carried out by placing the loaded composite in a medium devoid of protein and modulating the temperature to trigger the collapse of the hydrogel. This is coupled with collection of the release medium at specific time intervals. The protein content of the release medium is quantified by Bradford assay. This analysis enables a time based history of the protein released from the composite, which is simultaneously correlated to temperature and the state of the composite.
The mechanism of RelB dimerization
Thomas Ng, Don Vu, De-Bin Huang, Amanda Fusco and Gourisankar Ghosh Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA, 92093The Nuclear Factor-kappaB (NF-.B) family of dimeric transcription factors regulates the expression of several genes involved in cell physiology. There are five known NF-.B proteins, p50(NF-.B1), p52(NF-.B2), p65(RelA), c-Rel and RelB, that exist as homo- and heterodimers. Unlike other family members that form all possible functional combinatorial dimers, RelB forms heterodimers with only p100/p52 and p105/p50. Each NF-.B target gene is regulated by a subset of NF-.B dimers. The focus of this study is to understand the mechanism of restricted NF-.B dimer formation by RelB. The X-ray crystal structure of the RelB dimerization domain (DD) forms an intertwined homodimer. Based on the structures and the sequence of RelB and other dimers, we speculated certain residues are critical regulators of RelB dimer formation. We have shown that RelB forms an intertwined homodimer in solution as well. I have made several RelB mutants and tested their ability to form heterodimers with p52 using recombinant proteins using affinity based pull down experiments. Results of my experiments are described in my poster.
The Utility of N-Acylindole as a Versatile Synthetic Intermediate
Brian Nguyen, Matthew J. Buller, and Yoshihisa Kobayashi Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093Lactam is ubiquitous among heterocyclic natural products. A variation of the pyroglutamic acid was synthesized from an Ugi reaction of .-ketoacid with a convertible isonitrile, indole-isonitrile developed in our laboratory.1,2 The following .-lactam anilide was cyclized into the N-acylindole. A series of derivatives were synthesized from the N-acylindole. The carboxylic acid, amide, ester, aldehyde, and thioester were all substituted into the indole position in good yields under mild conditions. This methodology can be useful in making intermediates for natural product synthesis. Further studies with the Fukuyama reduction on the thioester of enantiomerically pure cysteine could prove to be a useful method to do a resolution and obtain separate diastereomers.
The Interactions of an apoE Peptide with Heparin and LDL Receptor-Related Protein
J. M. Ostrem, J. E. Croy, M. Guttman, and E. A. Komives Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA, 92093The low-density lipoprotein receptor-related protein (LRP) is a cell surface receptor responsible for clearing some 30 known ligands, one of which is apolipoprotein E (apoE) enriched .-VLDL particles. Cell surface heparan sulfate proteoglycans play a role in mediating this interaction and have been shown to internalize these particles through an LRP-independent pathway. A peptide from the N-terminal domain of apoE, apoE(130-149) interacts with each of the low-density lipoprotein receptor (LDL-R) homology domains (sLRPs 2, 3, and 4) of the LRP. Circular dichroism indicated that the apoE peptide is .-helical in the absence of lipid. Mutations at Lys 143 to Ala and Leu 144 to Pro caused a 90% loss of binding. The aliphatic residues in the apoE(130-149) peptide were assigned using homonuclear NMR methods. In order to complete the assignments of this small apoE mimetic peptide, and to perform chemical shift perturbation experiments, we have expressed the peptide behind the Trp leader sequence in E. coli. Results from NMR and circular dichroism studies of the peptide will be presented.
The Diffusion of Small Molecules into Flavoproteins as Revealed by Enhanced-Diffusion Molecular Dynamics Simulations
Conor Riley, Riccardo Baron, J. Andrew McCammon Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA, 92093Oxygen and other molecules of similar size have a fundamental role in a variety of enzymatic reactions carried out in living organisms. It is crucial to understand how these small molecules diffuse into proteins depending on global dynamics and local conformational changes. For example, no clear correspondence between structure and function has been proved to date to enable prediction on whether or how flavin-dependent enzymes react with oxygen [1-3]. We developed and applied a new computational strategy in order to enhance the sampling of molecular oxygen diffusion into proteins [4]. The methodology adopted enforces protein dynamics as close as possible to the physiological conditions, preserves the dynamical and structural role of protein motion (i.e. no restrained is applied), describes the role of water in atomistic detail by using explicit solvent molecular dynamics (MD) simulations. The results are described in the context of flavoproteins and provide a dynamic picture of diffusion otherwise invisible to experiments.
Purification and Characterization of Derlin-1
Jay Shen and Partho Ghosh Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA, 92093The endoplasmic reticulum associated protein degradation (ERAD) pathway is essential to cellular protein quality control, but the mechanisms by which the components are governed, is not entirely clear. We have developed a method to characterize one of the vital ER membrane proteins, Der1p, which recognizes misfolded proteins in the ERAD system. This method involves fusing Der1p to Mistic, a B. subtilis protein, increasing membrane protein expression. Following expression, our results from SDS-PAGE and immunodetection show that we have successfully solubilized and purified the Mistic-Der1p fusion protein via detergents and affinity chromatography. Based on these results, we conclude that the Mistic-Der1p fusion protein is produced in high yield and may be ready for structural characterization through crystallization and x-ray crystallography.
I.K. binding by I.K.
Eriko Shimada, Kim Huynh, Devin Lee Drew, Simon Bergqvist, Gourisankar Ghosh Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Dr, La Jolla, CA, 92093I.B Kinase (I.K) is a large protein complex formed by the subunits I.K., I.K., and I.K.. This kinase is a critical transducer of the upstream signal to the transcription factor, nuclear factor .B. In the I.K complex, I.K. interacts with both I.K. and I.K., and I.K. uses its C-terminus to bind to I.K.. The focus of this work is the quantitative and qualitative analysis of the I.K./I.K. complex. Using pure recombinant proteins we show that the minimal I.K. binding region of I.K. spans from residues 40 to 130. We also showed that the affinity of this complex is ~25nM. Finally, our results allows us to estimate that the basic unit of the I.K./I.K. complex requires a dimer of I.K. and a dimer of I.K..
Modification of a catalytic RNA by charged amino acids
Junaid Spall, Peter E. Struss and Ulrich F. M Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA, 92093According to the RNA world hypothesis, early forms of life on earth went through a stage called the RNA world. In this stage, RNA served both as catalyst (ribozyme) and genome, fulfilling the functions that are today mostly provided by proteins (catalysis) and DNA (genome). Our long-term aim is to recapitulate such an RNA world in vitro. There are three important components for an RNA world: a polymerase ribozyme, self-replicating membrane compartments and ribozymes that are capable of performing basic metabolic processes. A ribozyme has been developed which catalyze RNA polymerization. However, the longest primer extension achieved thus far is 14 nucleotides, whereas the ribozyme is 189 nucleotides long. Therefore, the ribozyme efficiency is far too low for self-replication. The limiting factor for polymerization efficiency was found to be low substrate binding affinity. The aim of this study is to improve the polymerization efficiency, by increasing the substrate binding affinity. We are trying to improve this interaction by coupling the polymerase ribozyme to positively charged amino acids, which might reduce the repulsion of the two negatively charged RNAs. To do that, we are currently trying to link arginine, histidine and lysine to amino-modified RNA by peptide coupling chemistry.
Applications of .Click Chemistry. in the Synthesis of Single Molecule Magnets
Casey J. Stephenson, Christopher C. Beedle, David N. Hendrickson Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093The labile-nature of first row transition metals employed in the synthesis of single molecule magnets (SMMs) has both positive and negative effects. On one hand, ease of ligand exchange leads to a wider array of complexes and the ability to tune their magnetic properties. However, this characteristic poses a synthetic challenge in that the outcome of these reactions is often difficult to control. Through click chemistry, a concerted 3-2 cycloaddition reaction between an azide and an ethynyl group, we hope to gain some control by making covalent linkages between magnetic clusters and with molecules such as C60. We are synthesizing 4-azido and 4-ethynyl derivatives of 2-hydroxymethylpyridine, a ligand used in the synthesis of many SMMs. We are also making ethynyl derivatives of C60 and ferrocene and a diazide derivative of 1,5-diaminonapthalene. Click chemistry might be also be applicable in the synthesis of single chain magnets resulting in 1, 2 or 3-dimensional networks of SMMs.
New luminescent dipyrrinato complexes
Van Sara Thoi Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093Metal-organic frameworks (MOFs) have been a recent topic of interest because they can be utilized as materials for molecular storage, sensors, and catalysis. Various studies have reported applications of MOFs in carbon dioxide and hydrogen storage. The functionality of MOFs will be greatly enhanced by the ability to fluoresce. This enhancement may be accomplished by the use of fluorescent .metalloligands.. Bis-(5-mesityldipyrrinato)zinc complex has been shown to be fluorescent (JACS 2004, 126, 2664-2665.) This study focuses on forming MOFs using three-fold symmetrical dipyrrinato complexes. Group 13 metals with a 3+ oxidation state have closed-shell electron configurations that also results in the formation of luminescent dipyrrin complexes. Tris-(5-mesityldipyrrinato)gallium and tris-(5-mesityldipyrrinato)indium were synthesized and shown to fluoresce at 365 nm excitation with a green emission centered at 528 nm. Further studies will be focused on the synthesis of MOFs using analogs of dipyrrin-based metalloligands.
Defining the Functions of a Prokaryotic Ubiquitin System
Sara Weitz and Pieter C .Dorrestein Department of Chemistry and Biochemistry Skaggs School of Pharmacy, University of California, San Diego, La Jolla, CA, 92093In eukaryotic cells, ubiquitin and ubiquitin-like proteins are important in signal transduction and degradation of proteins. A ubiquitin-like system has been identified in prokaryotic cells. Successful over-expression and purification of the prokaryotic ubiquitin and E1-type protein in greater than 90% purity has been achieved. It also has been shown, by FT-ICRMS, that the ubiquitin-like protein can be adenylated by the E1 protein and ATP (Fig. 1).
Discovery of New .B Binding Sites
Linda Yu, Vivien Wang and Gourisankar Ghosh Department of Chemistry and Biochemistry, University of California at San Diego, 9500 Gilman Dr, La Jolla, CA, 92093The NF-.B constitute a family of transcription factos that regulate the expression of a large number of genes by binding to specific DNA sequences known as .B sites. .B sites are consensus sequences of high G-C content. Many NF-.B target genes have multiple .B sites that vary in distance and sequence from each other. Regulation of the expression of some of these target genes has been studide and in all cases NF-.B alone is sufficient to regulate the gene expression. Database searches reveal that some of the promoters have sequences with loose .B consensus may function as an NF-.B binding site. We choose to test some of these loose .B sequences which are located near a consensus .B site. Our hypothesis is that the new .B-like sequence may bind NF-.B by cooperating with the neighboring consensus .B site. Our target promoters include ICAM-1, CD105, Apo3, GSTP1, and DEFB4. We have constructed luciferase reporter vectors, which allow the expression of the luciferase gene by these minimal target promoters. These minimal promoters contain DNA fragments bracketing only the potential .B sites that eliminates the contribution of the flanking sequences. HEK293T cells were co-transfected with different NF-.B subunits along with the reporter plasmid.
Phosphopantetheinylation of AcpC, a Protein on the Hemolytic Molecule Produced by Group B Streptococcus.
Wei H. Zheng, Pieter C. Dorrestein Department of Chemistry and Biochemistry, University of California at San Diego, 9500 Gilman Dr, La Jolla, CA, 92093The cyl cluster from Group B Streptococcus is responsible for the production of hemolytic toxin required for virulence. Using high-resolution mass spectrometry, we have shown that CylK, one of the 12 gene products of the cyl operon is a phosphopantetheinyl transferase that can activate the acyl carrier protein, AcpC. But due to the poor solubility of CylK we began searching for other phosphopantetheinyl transferases that can activate AcpC. We established that Sfp is another phopshopantetheinyl transferase that can load AcpC with CoA.