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  • Undergraduate Poster Abstracts
  • Biochemistry/Biophysics

    FRI-601 LIMITING THE GLYCATION OF HUMAN SERUM ALBUMIN WITH OMEGA-6 FATTY ACIDS

    • Richard Lauman III ;
    • Raymond Esquerra ;

    FRI-601

    LIMITING THE GLYCATION OF HUMAN SERUM ALBUMIN WITH OMEGA-6 FATTY ACIDS

    Richard Lauman III, Raymond Esquerra.

    San Francisco State University, San Francisco, CA.

    In 2012, roughly 29 million Americans were diagnosed with diabetes. Diabetes results in much morbidity and mortality. It was the 7th leading cause of death in 2013. Hyperglycemia is considered to be the principal cause of diabetic complications with many of its deleterious health consequences attributable to increased amounts of glycated proteins and their oxidative products: advanced glycation end products or AGEs. Understanding the chemistry controlling glycation is crucial in discovering the molecular mechanism behind the course of diabetic complications, and in developing treatments and therapies. Our hypothesis is that fatty acid binding to human serum albumin restricts glycation and AGE formation. Human serum albumin was incubated at a concentration of 30 g/L at a 15 molar ratio excess concentration against linoleate (C 18:2), oleate (C 18:1), palmitate (C 16:0), stearate (C 18:0), and a control sample. The samples were dialyzed against 50 mM glucose at 37 C for 48, 96, and 168 hours. Tryptophan fluorescence measured change in the hydrophobic pocket through the peak shift and decreased intensity of the emission. The quantification of fructosamines was measured by performing a nitro-blue tetrazolium assay. The samples were digested and measured via tandem mass spectrometry to determine the positions of the glycated lysines and arginines relative to the fatty acids. Preliminary data shows the addition of fatty acids limits the glycation of human serum albumin with the greatest margin of glycation prevention by stearate and palmitate. These findings could significantly aid in the development of therapies and treatments for diabetes.

    FRI-621 EXPLORING IRVING GEIS' LEGACY: AN OPEN-ACCESS EDUCATIONAL RESOURCE FOR STRUCTURAL BIOLOGY

    • Alex Alvarado ;
    • Nicole Werpachowski ;
    • Christine Zardecki ;
    • Stephen Burley ;

    FRI-621

    EXPLORING IRVING GEIS' LEGACY: AN OPEN-ACCESS EDUCATIONAL RESOURCE FOR STRUCTURAL BIOLOGY

    Alex Alvarado1, Nicole Werpachowski2, Christine Zardecki3, Stephen Burley3.

    1University of Southern California, Bakersfield, CA, 2Fordham University, New York, NY, 3Rutgers, The State University of New Jersey, New Brunswick, NJ.

    Prior to the advent of computer graphics and visualization technology, the process of visualizing protein and macromolecular structures at the level of the individual atom was very difficult. Starting with his work for Scientific American in the 1960's, world-renowned artist Irving Geis was able to take complex macromolecular information and present it in a way that enabled understanding by the general public. In collaboration with the Howard Hughes Medical Institute and the Geis family, the RCSB Protein Data Bank( RCSB PDB; rcsb.org) is creating a digital archive of Irving Geis' legacy. This new archive will be hosted within PDB 101, an educational website within the RCSB PDB scientific resource for structural biology. This digital archive, in the same way as Geis' work, will serve to enhance the understanding of structural biology for audiences new to Geis' artwork, particularly those with little or no experience. To enhance its impact, the archive will be integrated with interactive and modern molecular visualizations and connections to primary PDB data. The archive will also make Geis' images available for noncommercial usage.

    THU-619 METABOLIC PROFILES IN A TRANSGENDER ADOLESCENT POPULATION RECEIVING HORMONE THERAPY: A PILOT STUDY

    • Leslie Anne Villanueva ;
    • John Barth ;
    • Janie Benoit ;
    • Lee Ann Conard ;
    • Gylynthia Trotman ;

    THU-619

    METABOLIC PROFILES IN A TRANSGENDER ADOLESCENT POPULATION RECEIVING HORMONE THERAPY: A PILOT STUDY

    Leslie Anne Villanueva1, John Barth2, Janie Benoit3, Lee Ann Conard3, Gylynthia Trotman3.

    1University of Honolulu, Honolulu, HI, 2James M. Anderson Center for Health System Excellence, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, 3Cincinnati Children's Hospital Medical Center, Cincinnati, OH.

    The past decade has shown increasing numbers of transgender youth presenting for care and desiring initiation of gender-affirming sex steroid hormone therapy with estrogen or testosterone. Limited studies in the adult literature have suggested that sex hormones may lead to a change in the metabolic profile of a patient. This study aims to identify patterns in the lipid profiles and other metabolic markers of adolescent, transgender patients undergoing gender-affirming hormone treatment. We conducted a retrospective, 2008 to 2015, chart review examining the database of patients identified with gender dysphoria and who initiated sex steroid hormone therapy. We also reviewed baseline values and subsequent trends in BMI and BP in addition to biologic markers, including cholesterol levels (total cholesterol, LDL, HDL, and triglyceride), total testosterone level, estradiol levels, liver function tests, prolactin levels, HgbA1c, CBC, and electrolytes. A total of 222 charts were reviewed, of which 38 patients were included in the final analysis. There were significant changes in BMI and systolic BP between male to female (MTF) and female to male (FTM) patients after initiating gender-affirming hormones. FTM patients were noted to have a trend toward an increase in BMI (0.295 ± 0.468) and systolic BP (1.185 ± 4.133) while MTF patients had a decrease in BMI (-0.016 ± 0.629) and an increase in systolic BP (0.349 ± 4.870). Our results underscore the importance in monitoring metabolic risks and support the need for larger-scale studies describing the changes in lipid and metabolic profiles among the adolescent population undergoing gender-affirming hormone therapy.

    THU-610 NOVEL INTERACTING PROTEINS OF THE HEREDITARY SPASTIC PARAPLEGIA TYPES SPG11 AND SPG15 GENE PRODUCTS

    • Randall Hughes Jr. ;
    • Julia Stadler ;
    • Craig Blackstone ;

    THU-610

    NOVEL INTERACTING PROTEINS OF THE HEREDITARY SPASTIC PARAPLEGIA TYPES SPG11 AND SPG15 GENE PRODUCTS

    Randall Hughes Jr., Julia Stadler, Craig Blackstone.

    National Institutes of Health, Bethesda, MD.

    Hereditary spastic paraplegias are a genetically diverse group of disorders (SPG1-73) with the common feature of lower extremity spasticity and weakness. Inheritances include autosomal dominant, recessive, and X-linked. SPG11 and SPG15 are the most common autosomal recessive forms with virtually identical clinical symptoms including thin corpus callosum, cognitive dysfunction, white-matter changes on MRI, ataxia, and retinopathy. The SPG11 and SPG15 proteins bind one another and are critical for autophagic lysosome reformation. To understand the regulation of SPG11/15-related processes, we studied their protein interactors, starting with proteomics data from another group, suggesting that Rab7 and Rab26 might bind SPG11/15. In fact, the Rab protein family has been known to regulate endosome and lysosome biogenesis and trafficking. Using coimmunoprecipitation (Co-IP) studies, we were able to confirm an interaction of the SPG15 protein with the endolysosomal Rab protein Rab7. We are now studying the effects of Rab7 functional alterations (e.g., GTPase activity) in autophagic lysosome reformation.

    THU-611 DETERMINING THE CRYSTAL STRUCTURE OF THE ANKYRIN REPEAT AND SOCS BOX PROTEIN ASB9 WITH CREATINE KINASE

    • Ashley Chong ;
    • Elizabeth Komives ;

    THU-611

    DETERMINING THE CRYSTAL STRUCTURE OF THE ANKYRIN REPEAT AND SOCS BOX PROTEIN ASB9 WITH CREATINE KINASE

    Ashley Chong, Elizabeth Komives.

    University of California, San Diego, La Jolla, CA.

    The ankyrin repeat and suppressor of cytokine signaling (SOCS) box, or ASB, is a family of proteins that act as the substrate identification subunits in cullin RING E3 ubiquitin ligases. These ligases transfer ubiquitin to protein complexes that are targeted for degradation by the proteasome. In particular, ASB9 protein associates with elongins B and C, RBX2, and cullin 5 to form a cullin RING E3 ligase. Binding assays, isothermal titration calorimetry, and hydrogen-deuterium exchange experiments demonstrated ASB9 binding activity toward brain-type creatine kinase (CKB). However, the interaction and binding of ASB with creatine kinase is still not well understood. Recent studies have suggested that ASB9 may be a biological indicator for certain cancers. Previously, we identified single molecules of ASB9 that bind dimers of CKB. Our objective is to determine the crystal structures of ASB9 with CKB by X-ray crystallography to understand the binding interface between the two proteins. Different constructs of ASB9 are being explored in order to form a stable complex containing CKB, ASB9, and elongins B and C for crystallization. Protein crystallization will also be carried out using the hanging-drop method as this has been hypothesized as being the most effective way of obtaining protein crystals. This will be conducted using Hampton screens on 24 well trays. It is expected that ASB9 will bind to a dimer of CKB as well as the SOCS box interacting with elongin B and C. These structures will expand upon the understanding of ubiquitin ligation by RING E3 ligases.

    THU-620 NONSPECIFIC ENVIRONMENTAL STRESSES AND THEIR EFFECTS ON EMBRYONIC DEVELOPMENT OF DROSOPHILA MELANOGASTER

    • Dihae G.Yook ;
    • Monica Lacy ;

    THU-620

    NONSPECIFIC ENVIRONMENTAL STRESSES AND THEIR EFFECTS ON EMBRYONIC DEVELOPMENT OF DROSOPHILA MELANOGASTER

    Dihae G.Yook, Monica Lacy.

    Vanderbilt University, Nashville, TN.

    Nonspecific stresses such as heat stress, ultraviolet radiation, and hypoxic conditions applied to Drosophila melanogaster embryos at the onset of gastrulation causes defects later in embryonic development. In heat stress conditions, potential premature cellular death can be a proponent to defects during embryonic development of Drosophila. It is hypothesized that an applied heat shock will result in premature apoptotic events that can lead to holes in the amnioserosa. Heat stress was applied by placing gastrulating Drosophila embryos in a 38 °C water bath for 30 minutes. Ultra-violet radiation stress and hypoxic stress were 2 additional stresses applied to Drosophila embryos. Ultra-violet radiation was applied at 1.7 nJ for 2 minutes, and hypoxic stress was applied for 30 minutes. Embryos were allowed to recover post shock. In conclusion, there were several defects, but the most prevalent defects were holes in the amnioserosa, failure in germband retraction, failure in germband extension, and head defects in the Drosophila melanogaster embryo in all stress-induced conditions.

    THU-617 AN EXAMINATION OF FERRIC IRON AND NITRATE REDUCTION ACTIVITY IN ACIDOBACTERIUM CAPSULATUM

    • Leandrew Dailey ;
    • Leslie Sommerville ;

    THU-617

    AN EXAMINATION OF FERRIC IRON AND NITRATE REDUCTION ACTIVITY IN ACIDOBACTERIUM CAPSULATUM

    Leandrew Dailey, Leslie Sommerville.

    Fort Lewis College, Durango, CO.

    Acidobacterium capsulatum is an acidophilic, heterotrophic, aerobe ubiquitous to soils and aquatic environments throughout the world. It is capable of growing on glucose as its sole carbon source. Comparative studies of Acidobacteria genomes suggests A. capsulatum may be capable of reducing Fe(III) and nitrite, while other Acidobacteria may be able to reduce nitrate. Additional in situ studies showed iron reduction capabilities in Acidobacteria is prevalent. The ability to reduce Fe(III), nitrite, and nitrate allows these organisms to potentially play a significant role in carbon, nitrogen, and iron cycling in soil and aquatic environments. In order to test this hypothesis and better understand these capabilities, A. capsulatum was grown in defined media containing an insoluble Fe(III) substrate, goethite (FeOOH), or KNO3 for several days, with glucose as the sole carbon source. Ferrozine was used as the indicator of Fe(II) in solution, and protein concentration was used as a measure of bacterial growth. Under these conditions, it was confirmed that A. capsulatum can couple iron reduction to cell growth and lacks the ability to reduce nitrate. Surprisingly, A. capsulatum growth was completely inhibited with 1.0% potassium nitrate. Future studies will continue to define the relationship between iron reduction, nitrate toxicity, growth, and glucose consumption. Other studies will focus on the putative nitrite-reducing gene (NirBD) in A. capsulatum to determine whether this is for dissimilatory or assimilatory nitrite reduction. This study will enable us to better understand A. capsulatum’s ability to grow in soil and provide a crucial role in soil maintenance.

    THU-613 MODULATING KINESIN-1 AFFINITY FOR MICROTUBULES

    • Ramona Luna ;
    • Michael Vershinin ;
    • Jared Bergman ;

    THU-613

    MODULATING KINESIN-1 AFFINITY FOR MICROTUBULES

    Ramona Luna1, Michael Vershinin2, Jared Bergman2.

    1University of Texas Rio Grande Valley, Brownsville, TX, 2The University of Utah, Salt Lake City, UT.

    Cargo transport along cytoskeletal filaments is crucial for cellular performance. Of particular interest are microtubules: filaments in the cytoskeleton of eukaryotic cells that serve as the highway system. These filaments are formed by alpha and beta tubulin dimers which polymerize into a tubular structure only 25 nm in diameter. Kinesin-1 is a molecular motor that binds microtubules and hydrolyzes ATP to move processively for hundreds and even thousands of nanometer along these filaments. Synthesizing artificial structures that support sustained molecular transport has recently become an area of acute interest in research. Beta tubulin has a helix known as H12, which is a major binding site for kinesin-1. Short peptides with helical structure similar to endogenous H12 were prepared in our lab. We then used optical trapping to study kinesin-1 affinity for microtubules and compare the kinesin-microtubule binding affinities for samples with and without H12 mimicking peptide. These competitive binding assays help us determine how well the peptides bind to kinesin and whether this binding correlates with the helicity of the peptides.

    FRI-617 HIGH-RESOLUTION VISUALIZATION OF THE PARMRC COMPLEX WITH THE AID OF DNA ORIGAMI STRUCTURES

    • Kate Arriola ;
    • Tural Aksel ;

    FRI-617

    HIGH-RESOLUTION VISUALIZATION OF THE PARMRC COMPLEX WITH THE AID OF DNA ORIGAMI STRUCTURES

    Kate Arriola1, Tural Aksel2.

    1Harvey Mudd College, Claremont, CA, 2University of California, San Francisco, San Francisco, CA.

    Common to all living species, cell division provides a means of creating genetically identical daughter cells from a progenitor. To maintain high fidelity of the daughter cells, it is crucial that duplicated genetic material is evenly segregated between dividing cells. Bacteria have a mechanism involving a complex called ParMRC which segregates genetic material called plasmid. ParMRC comprises an actin-like protein called ParM, a DNA-binding adaptor protein called ParR, and a DNA fragment called parC that is located in the plasmid. Research has shown that ParR binds to parC, and one ParR-parC complex binds each end of the ParM filament, driving ParM elongation and eventually leading to segregation of plasmids to daughter cells. However, the structural mechanism by which ParR-parC binding initiates ParM elongation remains unknown. Previous attempts to determine the high-resolution structure of ParMRC have failed due to the complex’s dynamic nature. Fortunately, cryo-EM is a promising technique for high-resolution structure determination of dynamic systems such as actin filaments and microtubules. However, cryo-EM applications have been limited to macromolecules larger than 200 kDa. Here, we use DNA nanotechnology to design a DNA origami structure, 5 MDa in size, as a fiducial marker to visualize ParMRC (~150 kDa) using cryo-EM. The DNA structure resembles a hinge and assumes a closed conformation upon complexing with ParMRC. Preliminary results indicate that the hinge is properly folded and incorporates the parC-containing DNA substrate required for ParMRC assembly. The hinge’s flexibility should accommodate different substrates thus permitting easy adaptation for studying other DNA-binding proteins.

    FRI-610 PURIFICATION AND ANALYSIS OF HUMAN LIVER FATTY ACID BINDING PROTEIN (FABP1)

    • Charlene Valdez ;
    • Ann Hertzel ;
    • Michael Downey ;
    • David A. Bernlohr ;

    FRI-610

    PURIFICATION AND ANALYSIS OF HUMAN LIVER FATTY ACID BINDING PROTEIN (FABP1)

    Charlene Valdez1, Ann Hertzel2, Michael Downey2, David A. Bernlohr2.

    1Trinity Washington University, Washington, DC, 2University of Minnesota, Minneapolis, MN.

    Obesity is a chronic metabolic disorder affecting over one-third of Americans. It is characterized by excessive accumulation of adipose tissue and is correlated with increased risk for cardiovascular disease, hepatosteatosis, and type 2 diabetes. Human liver fatty acid binding protein (FABP1) functions to metabolize long chain fatty acids in the hepatocyte and is linked to metabolic processes such as lipid storage, oxidation, and gene expression. Whole-body ablation of FABP1 in high-fat fed obese C57Bl/6J mice results in reduced hepatosteatosis and increased insulin sensitivity. This suggests that small molecule inhibitors of FABP1 may be efficacious in reducing hepatic steatosis. To facilitate FABP1 inhibitor studies, the protein was expressed in E. coli and purified using nickel affinity column chromatography followed by delipidation via lipidex column chromatography. X-ray crystallographic studies have revealed that the lipid binding domain of FABP1 is found within a large interior cavity. Lipid binding was measured using the fluorescent fatty acid analogue 1,8-ANS and revealed a binding affinity of ~1.8 μM. Moreover, fatty acids displaced 1,8-ANS from the binding cavity, thereby enabling analysis of therapeutic ligands. These studies offer the potential for identifying small molecule inhibitors of LFABP, useful in the prevention of liver steatosis. (Supported by the NIH R25 HL088728 and the University of Minnesota Life Science Summer Undergraduate Research Program.)

    THU-623 FREE ENERGY CALCULATION OF GLYCOPHORIN-A USING HYBRID STEERED MOLECULAR DYNAMICS

    • Lawrence Rhoads ;
    • Liao Chen ;

    THU-623

    FREE ENERGY CALCULATION OF GLYCOPHORIN-A USING HYBRID STEERED MOLECULAR DYNAMICS

    Lawrence Rhoads, Liao Chen.

    The University of Texas at San Antonio, San Antonio, TX.

    Computer simulations will someday allow us to run biological experiments via simulations rather than use actual biological material. However, for now, our knowledge of the basic structure of biomolecules and how they interact is too limited to use simulations for very complicated biological processes. We can, however, use current computational modeling techniques to study biological systems that are simple and provide important qualitative and quantitative information that experimentalists can use to guide the direction of their experiments. We propose to use computer simulation techniques to understand the association between glycohphorin-A proteins within a membrane environment, which could lead to an understanding of how glycophorin-A interacts with other biological molecules like viruses, such as influenza, and drug molecules. We will use a novel computer modeling technique, called hybrid steered molecular dynamics (hSTMD), to model a glycophorin-glycophorin complex in both a saline and membrane (bilipid layer) environment. We hypothesize that the binding energy of the complex in the membrane will be stronger than in a saline environment. This information will be important in understanding how glycophorin-A interacts with its membrane environment, which in turn will provide insight into how the protein might interact with other biomolecules. In addition, this study will prove the feasibility of hSTMD in protein-protein simulation studies and perhaps, in the future, aid experimentalists with the interpretation and analysis of their free energy experiments dealing with such complexes.

    THU-609 CREATING COMPUTATIONAL MODELS FOR PROTEIN ASSEMBLY

    • Alejandro Herrera ;
    • Sagar Khare ;
    • Christopher Herrera ;
    • Denzel Zhu ;

    THU-609

    CREATING COMPUTATIONAL MODELS FOR PROTEIN ASSEMBLY

    Alejandro Herrera, Sagar Khare, Christopher Herrera, Denzel Zhu.

    Rutgers University, New Brunswick, NJ.

    Binding domains are a type of protein domain that binds to a specific molecule. Binding domains can be inserted onto proteins. Proteins with binding domains can be linked together to form protein assemblies. Assemblies have been shown to improve reaction rates, reduce transit time of intermediates, and can reduce diffusion of toxic intermediates. While protein assemblies prove to be beneficial, inserting domains can reduce the stability and function of the protein. There are many locations for domain insertion, some that would result in a useless protein and others that would result in a successful assembly. In addition, there are many different linkers that can be inserted for domain linkers. By creating computational models for domain insertion, we can filter out assemblies that are unlikely to come together. The proteins modeled were green fluorescent protein (GFP) and red fluorescent protein (RFP). Using protein data bank files from the Research Collaboratory for Structural Bioinformatics, we created models for GFP and RFP assemblies. The assemblies were created using the programs PyMOL and Rosetta. Criteria such as energy score and angle of proteins were used to filter out preferred models. Experimental validation is being investigated.

    FRI-615 ALTERED ELECTRON TRANSFER KINETICS IN NATURALLY NOT OCCURRING 2C8 POLYMORPHIC VARIANTS

    • Kimberly Sam ;
    • William Arnold ;
    • Susan Zelasko ;
    • Daryl Meling ;
    • Aditi Das ;

    FRI-615

    ALTERED ELECTRON TRANSFER KINETICS IN NATURALLY NOT OCCURRING 2C8 POLYMORPHIC VARIANTS

    Kimberly Sam, William Arnold, Susan Zelasko, BS1, Daryl Meling, Aditi Das.

    University of Illinois, Urbana-Champaign, Urbana, IL.

    Cytochrome P450 2C8 (CYP 2C8) is an extrahepatic enzyme that plays a significant role in the metabolism of over 20% of clinically relevant drugs, including paclitaxel, an anticancer drug used to treat ovarian, breast, and lung cancers, as well as Kaposi sarcomas. It has been noted that naturally occurring polymorphic variants CYP 2C8*2 and CYP 2C8*3 have prevalence in African American and Caucasian populations at 18% (*2) and 13% (*3) respectively. It is believed that the mutations lie within the putative binding interface between CYP 2C8 and its redox partner, cytochrome P450 reductase (CPR). The location of these mutations plays a critical role in the efficiency of the protein, as previous studies have shown reduced substrate hydroxylation due to inefficient use of electrons toward the production of reactive oxygen species. To quantify the inefficient use of electrons, we measured the amount of hydrogen peroxide formed using nanodisc technology to study membrane protein function in the native-like bilayer environment. Additionally, the electron transfer rates between the CYP 2C8 variants and CPR were determined through stopped-flow studies to better understand how these mutations alter electron flow. Hydrogen peroxide production and altered electron transfer rates were observed in both CYP 2C8*2 and CYP 2C8*3 in comparison to the wildtype, demonstrating that these mutations have a significant effect on the enzyme efficiency. The results of these studies propose therapeutic potentials that will allow for the development of drugs that better fit populations carrying these variants.

    FRI-608 INFLUENCE OF ENVIRONMENTAL CHANGES IN THE GENE EXPRESSION OF BACILLUS SUBTILIS

    • Ilyana Martinez Cosme ;
    • Siddarth Srinivasan ;
    • Shmuel Rubinstein ;

    FRI-608

    INFLUENCE OF ENVIRONMENTAL CHANGES IN THE GENE EXPRESSION OF BACILLUS SUBTILIS

    Ilyana Martinez Cosme1, Siddarth Srinivasan2, Shmuel Rubinstein2.

    1Universidad del Turabo, Gurabo, PR, 2Harvard University, Cambridge, MA.

    Bacillus subtilis is a bacterium used as a model system to study swarming, biofilm formation, and sporulation. It is important because it exhibits a sophisticated, self-organized, and cooperative behavior. We focused on understanding the influence of different environmental growth conditions on gene expression and growth pattern of the B. subtilis. For our bacterial model, we first inoculated small Petri dishes containing the biofilm-promoting nutritive medium MSgg (minimal salts glycerol glutamate) gelified with agarose. Using a live cell incubator box and confocal microscope we perform wide-field time-lapse microscopy at the edge of the biofilm, to follow the evolution of our cells and observe the growth patterns and gene expression of the bacteria. At low agar concentration (0.5 wt%), the cells exhibit a swarming behavior, characterized by a mono-layer of predominantly motile bacteria that forms an unstable front, which gives rise to fingering and branching clockwise growth patterns. In higher agar concentration (> 1.5 wt%), a tendril forming and sliding mode is observed, where the growth at the edges is dominated by matrix/chaining bacteria. We characterized the ratio of motile and matrix producers as it encountered different environmental conditions.

    THU-604 EXPLORING DOMAIN COMMUNICATION IN HUMAN CYSTATHIONINE Œ ≤ -SYNTHASE

    • Bushra Bibi ;
    • Dana Kennedy ;
    • Raymond Esquerra ;

    THU-604

    EXPLORING DOMAIN COMMUNICATION IN HUMAN CYSTATHIONINE Œ ≤ -SYNTHASE

    Bushra Bibi, Dana Kennedy, Raymond Esquerra.

    San Francisco State University, San Francisco, CA.

    Human cystathionine β-synthase (CBS) is a pyridoxal phosphate-dependent (PLP) enzyme that contains heme and catalyzes the β-replacement reactions of serine by homocysteine to give cystathionine. Mutation in this key mammalian enzyme causes hyperhomocysteinemia, which results in aggressive arterial diseases, a clinical phenotype observed in patients. Although the role of PLP is similar to other PLP-dependent enzymes that catalyze the exchange of the hydroxyl group of serine to thiolate in homocysteine, little is known about the role heme plays in regulating the catalytic process. The overall goal of this work is to use time-resolved absorption and circular dichroism spectroscopy to integrate how small ligand binding to the heme induces confirmation changes at the PLP active site in CBS. In this work, we present a coupled spectrophotometric assay to monitor CBS kinetics. Cystathionine produced from CBS undergoes oxidative deamination by L-amino acid oxidase to yield hydrogen peroxide, which is detected colormetrically using 2, 2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) and horseradish peroxidase. We also use natural and magnetic circular dichroism spectroscopy to examine how the binding of small gasotransmitters (CO, NO, and H2S) affect the PLP active site and heme binding domain. Interactions between the heme and PLP active site are also probed using time-resolved absorption spectroscopy following ligand photolysis. The outcome of the proposed work will employ CBS efficiency to unravel the mechanism associated with heme-active site interactions and provide evidence for a ligand specific regulation mechanism in heme-dependent cystathionine β-synthase.

    FRI-612 MUTAGENIC ANALYSIS OF THE RBFOX1 C-TERMINUS TO DETERMINE ITS ROLE IN PROTEIN COMPLEX ASSEMBLY AND SPLICING REGULATION

    • Robert Vasquez Jr. ;
    • Douglas Black ;

    FRI-612

    MUTAGENIC ANALYSIS OF THE RBFOX1 C-TERMINUS TO DETERMINE ITS ROLE IN PROTEIN COMPLEX ASSEMBLY AND SPLICING REGULATION

    Robert Vasquez Jr.1, Douglas Black2.

    1San Diego State University, San Diego, CA, 2University of California, Los Angeles, Los Angeles, CA.

    Alternative splicing is a central mechanism for regulating protein output that allows the majority of mammalian genes to produce multiple products through controlled changes in splice sites. One class of splicing regulators are the Rbfox proteins. The Black lab has recently learned that these proteins are associated with the chromatin pellet of fractionated nuclei and not soluble nucleoplasm as some other splicing regulators. Within this cellular compartment the Rbfox proteins are complexed with 8 other proteins of similar stoichiometry. This mulit-protein complex has been named the large assembly of splicing regulators (LASR). The data indicate that one Rbfox protein must assemble with the LASR complex to regulate splicing, and can alter the activity of other LASR components. We find that the c-terminal domain of Rbfox is required for its association with LASR. To refine our understanding of this interaction we are testing additional Rbfox mutations. By exchanging segments of deactivated mutant Rbfox with wild-type sequences in the c-terminus, we can elucidate the minimal region necessary for protein activation. Four segments of the c-terminus will be added back to the c-terminal deletion mutant to determine if they are sufficient to restore splicing. Techniques will include gateway cloning, culturing HEK293T cells, western blot analysis, and RT-PCR. Our findings will provide fundamental new understanding of the mechanisms of Rbfox dependent splicing.

    THU-605 INVESTIGATION OF THE STRUCTURE OF RECOMBINANTLY EXPRESSED INFLUENZA M1 MATRIX PROTEIN

    • Hector Caldera ;
    • Nancy Hom ;
    • Kelly Lee ;

    THU-605

    INVESTIGATION OF THE STRUCTURE OF RECOMBINANTLY EXPRESSED INFLUENZA M1 MATRIX PROTEIN

    Hector Caldera1, Nancy Hom2, Kelly Lee2.

    1Fort Lewis College, Durango, CO, 2University of Washington, Seattle, WA.

    Influenza M1 matrix protein plays a key role in the life cycle and proliferation of the virus. Understanding how the M1 protein assembles in order to influence cell entry, ribonucleoprotein (RNP) organization, genome packaging, and viral budding is important for potential future research in anti-viral drug design and development. Previous biochemical studies have uncovered some of the roles of M1 with no corresponding reports of detailed structural analysis of the full-length protein. In other investigations, truncated M1 proteins were used to determine crystal structures or low-resolution methods were used to probe the full-length protein’s organization. It remains unclear how the M1 protein assembles into the matrix protein shell, which envelops the internal RNP complexes. The goal of our current investigation is to elucidate the native assembly structure of full-length M1 matrix protein. We are recombinantly cloning and expressing the M1 protein of A/Udorn/1972 (H3N3) using E. coli bacterial cells. After purification, electron microscopy (EM) techniques will be used to image M1 protein assemblies. We will process the EM images and dock in the crystal structures of truncated M1 to generate a 3D model of the assembled M1 quaternary structure and gain insight into the molecular contacts of M1 protein. Additional molecular interactions between M1 protein, RNPs, the viral lipid membrane, and tail domains of surface proteins may be determined in the future. A better understanding of M1 could be helpful in designing potential drugs that disrupt M1’s function thus disrupting the life cycle of influenza.

    FRI-602 BIOREMEDIATION OF LEAD USING MODIFIED S-LAYER PROTEINS IN

    • Christopher De Alba ;
    • Elizabeth S. Bailey ;
    • Wesley L. Jenkins ;
    • Azariah C. Coblentz ;
    • Michael P. Olvera ;
    • Gillian V. Lui ;
    • Caroline M. Ajo-Franklin ;
    • Jenny A. Cappuccio ;

    FRI-602

    BIOREMEDIATION OF LEAD USING MODIFIED S-LAYER PROTEINS IN

    Christopher De Alba1, Elizabeth S. Bailey1, Wesley L. Jenkins1, Azariah C. Coblentz1, Michael P. Olvera1, Gillian V. Lui2, Caroline M. Ajo-Franklin3, Jenny A. Cappuccio1.

    1Humboldt State University, Arcata, CA, 2Stanford Woods Institute for the Environment, Stanford University, Stanford, CA, 3Molecular Foundry, Lawrence Berkeley National Lab, Berkeley, CA.

    Heavy metal contamination of soils and waterways due to industrial processes such as mining, continues to be a problem in the U.S. Recent reports indicate that microbial surface layers (S-layers) may be able to absorb heavy metals. Here we describe the design of engineered Caulobacter vibrioides for use in bioremediation. Caulobacter vibrioides possess an S-layer protein (RsaA) which forms a 2D crystalline array with hexagonal symmetry. This protein, which covers the outermost surface of the cell, could increase the capability of C. vibrioides to absorb heavy metals from the environment if modified. Our team has engineered various binding motifs into the RsaA S-layer protein of C. vibrioides and studied the effectiveness of Pb sequestration. Sequencing and S-Layer extraction has confirmed 2 of these strains and S-layer protein export in three strains. Growth studies in PYE media indicate that recombinant and wild-type strains can tolerate and grow in at least 25 ppm Pb in solution. Atomic absorption spectroscopy of growth media after 19 hours Pb exposure reveals that engineered C. vibrioides strains can remediate 25.00 and 50.00 ppm Pb to ~2 ppm Pb. Calculated Pb absorbed per cell mass for strains HCm 021, 027, and 028 in media + 25 ppm Pb were 197.78 ± 2.14, 231.32 ± 3.10, and 168.69 ± 22.65 ppm/g respectively and 90.55 ± 1.07 ppm/g for the wildtype. These results indicate that Pb is sequestered preferentially in our engineered strains. Future goals of this project include determining binding constants of lead to cells, binding of other heavy metals, and creation of a bioremediation filter.

    THU-607 MAGNESIUM FACILITATES CALMODULIN BINDING TO NEURONAL NITRIC OXIDE SYNTHASE

    • Lara Manimbao ;
    • Cameron Grant ;
    • Pooncharas Tipgunlakant ;
    • Raymond Esquerra ;

    THU-607

    MAGNESIUM FACILITATES CALMODULIN BINDING TO NEURONAL NITRIC OXIDE SYNTHASE

    Lara Manimbao, Cameron Grant, Pooncharas Tipgunlakant, Raymond Esquerra.

    San Francisco State University, San Francisco, CA.

    Neuronal nitric oxide synthase (nNOS) is a calcium Ca2+/calmodulin (CaM) dependent enzyme that generates nitric oxide from the amino acid arginine. Nitric oxide (NO) is responsible for numerous physiologically significant processes, including vasodilatation, angiogenesis, neuronal signaling, and cytotoxic immune response. Mg2+ plays many significant roles physiologically and magnesium therapies are widely implemented for the treatment of variety of ailments. However, the mechanism of action remains unresolved. This work examines the relationship between Mg2+ and neuronal nitric oxide synthase activation. Specifically, we demonstrate that the synergistic role Mg2+ cations play in facilitating the binding of calmodulin to nNOS. We also examine the effect of Mg2+ on binding of calmodulin to nNOS using fluorescence spectroscopy and affinity chromatography. We also use circular dichroism spectroscopy to show that Mg2+ can induce conformational changes in calmodulin in the absence and presence of calcium. This work indicates that both Mg2+ and Ca2+ levels may play an important physiological role in regulating nNOS activity.

    THU-612 DEVELOPING AN ENVIRONMENTAL STIMULI-INDUCED SYSTEM FOR PROTEIN PROXIMITY

    • Larisa Breden ;
    • Guihua Zeng ;
    • Roushu Zhang ;
    • Wei Wang ;
    • Fu-sen Liang ;

    THU-612

    DEVELOPING AN ENVIRONMENTAL STIMULI-INDUCED SYSTEM FOR PROTEIN PROXIMITY

    Larisa Breden, Guihua Zeng, Roushu Zhang, Wei Wang, Fu-sen Liang.

    The University of New Mexico, Albuquerque, NM.

    Elevated levels of Fe2+ and H2O2 have been marked in neurodegenerative brains of both Alzheimer’s disease (AD) and Parkinson’s disease (PD) patients, contributing to the pathogenesis. We hypothesize that a novel, synthetic, biology-based gene therapy strategy that incorporates environment-sensing and bio-computing logics (i.e., “AND” Boolean logic gate) can ensure a desired response is generated only when a specific environment exists. This system is based on 2 reported chemically induced proximity (CIP) systems: absicsic acid (ABA) and gibberellin (GA). The systems use exogenous chemical inducers to regulate biological processes via protein proximity. We chemically caged ABA and GA to be used as sensors for Fe2+ and H2O2 and as inducers for respective downstream cellular processes. We predict that when free ABA and/or GA is produced in the presence of Fe2+ and H2O2, the proteins will fuse to a DNA binding or transactivation domain. For the model system, this induces expression of a downstream protein, green florescence protein (GFP), which can later be replaced by a therapeutic protein. We plan to expose the engineered cells to concentrations of Fe2+ and H2O2, monitoring GFP expression with florescence microscopy. The detection of GFP fluorescence only in cells with both Fe2+ and H2O2 concentrations seen in AD and PD will indicate a condition-specific CIP system. By engineering an environment-detecting cell, we address a major issue of cell therapy development. We predict that our system can be modified to respond to the specific biological conditions of many diseases in addition to AD and PD.

    THU-600 THE ROLE OF CONSERVED LYSINES IN DNA POLYMERASE

    • Kalia Ostrander ;
    • Joeseph Dahl ;

    THU-600

    THE ROLE OF CONSERVED LYSINES IN DNA POLYMERASE

    Kalia Ostrander, Joeseph Dahl.

    University of California Santa Cruz, Santa Cruz, CA.

    DNA polymerases (DNAPs) are highly conserved enzymes that are tasked with faithful replication of the genome. Eukaryotic DNA synthesis is largely conducted by B-family DNAPs, which typically have 2 active sites: the polymerase active site where nucleotide incorporation occurs, and the exonuclease active site, where editing occurs. Using the B-family DNAP from the bacteriophage phi29 (phi29 DNAP), our collaborators have identified 2 highly conserved residues, Lys383 and Lys498. These residues were shown to have a direct connection to the catalytic balance between the 2 active sites. More recently, high resolution X-ray crystal structures have been determined showing the specific contacts of these residues. A single molecule technique has been established to describe the kinetic mechanisms that reside at the branch point between DNA synthesis and editing activities. The biophysical mechanisms that gave rise to the phenotypes observed in the established mutants will be determined by capturing individual complexes of mutant phi29 DNAP bound to DNA atop a nanopore. The behavior of the enzyme will be analyzed as a function of force (voltage) and nucleotide concentration, allowing the rates of primer strand transfer, DNA displacement, and nucleotide binding to be quantified. Preliminary data supports the previously published observations that mutants in these residues affect the balance between polymerase and exonuclease activities. Specifically, K383R has a decreased affinity for nucleotides and K498R shifts equilibrium towards the pre translocation state.

    THU-615 ENZYMATIC ACTIVITY AND PRELIMINARY CRYSTALLOGRAPHIC ANALYSIS OF A FUNGAL CATALASE

    • Asia Payne ;
    • M. Cristina Vega ;

    THU-615

    ENZYMATIC ACTIVITY AND PRELIMINARY CRYSTALLOGRAPHIC ANALYSIS OF A FUNGAL CATALASE

    Asia Payne1, M. Cristina Vega2.

    1Spelman College, Lakeland, TN, 2Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cientificas, Madrid, ES.

    Catalases are enzymes found in nearly all living organisms, from bacteria to fungi, plants or animals, exposed to oxygen. Catalases catalyze the decomposition of harmful hydrogen peroxide into water and oxygen, thereby protecting the cell from the oxidative damage caused by reactive oxygen species. Fungal and yeast enzymes are particularly interesting from the biotechnological and industrial perspectives, since they often exhibit robust catalytic and stability properties. Several fungal catalases have been structurally characterized by X-ray crystallography from organisms such as Penicillium vitale and Micrococcus lysodeikticus. More recently, the structure of Hansenula polymorpha catalase has been reported, but much remains to be known about the structure and activity of catalases from fungal sources. Pichia pastoris is a model methylotrophic yeast in peroxisomal research and biotechnology with applications ranging from food to the pharmaceutical industry. In order to fully understand the enzymatic activity and structure of the P. pastoris catalase (PpCAT1), we have cloned, overexpressed, and purified PpCAT1. To further our knowledge on the structure-function relationship of fungal catalases, we set out to crystallize PpCAT1 and determine its 3D structure by X-ray crystallography. Crystals were diffracted in the ALBA synchrotron light source. The best crystals diffracted to 2.35 Å resolution, and the complete structure determination is in progress. In conclusion, the enzymatic activity and structural results found will provide further biological knowledge about the PpCAT1 as well as provide information for future industrial applications.

    FRI-623 THE ANALYSIS OF AMINO ACID NEUROTRANSMITTERS IN C. ELEGANS USING MICROCHIP ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION

    • Vincent Fiorentino ;
    • Nathan Oborny ;

    FRI-623

    THE ANALYSIS OF AMINO ACID NEUROTRANSMITTERS IN C. ELEGANS USING MICROCHIP ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION

    Vincent Fiorentino, Nathan Oborny.

    The University of Kansas, Lawrence, KS.

    C. elegans is used to study various biological phenomena. Having had its entire genome sequenced allows for genetic manipulation enabling studies of numerous biological characteristics. Glutamic acid decarboxylase (GAD) converts glutamate to the neurotransmitter gamma-aminobutyric acid (GABA). The single gene encoding GAD is called unc-25 in C. elegans. Previous work suggests that mutations in unc-25 ablate the worm's ability to make GABA. The goal of this work is to validate that the unc-25 animals are entirely GABA free. Should GABA be detected, there would be evidence to support the existence of unknown alternative pathways for GABA production. Understanding the role and pathways for GABA in C. elegans is important in understanding its nervous system which mirrors higher organisms such as humans. Because GABA exists in such low concentrations, preliminary work has focused on other amino acid neurotransmitters that exist at higher concentrations: glutamate, aspartate, and glycine. By fluorescently tagging the desired amino acid neurotransmitters and using microchip electrophoresis (ME) with laser induced fluorescence detection (ME-LIF), one can separate and selectively detect with high sensitivity each targeted analyte. Starting with these 3 amino acids has permitted verification of the extraction method as well as the ME-LIF separation-based assay vis-a-vis their ratio of charge to hydrodynamic radius. Additionally the small channel size offered with ME allowed for the analysis of nanoliter-sized samples.

    THU-614 DISCOVERING NOVEL COMPOUNDS TO STABILIZE THE E2F-RETINOBLASTOMA PROTEIN COMPLEX IN THE CELL CYCLE

    • Elise Brown ;
    • Cameron Pye ;
    • Tyler Liban ;
    • Seth Rubin ;

    THU-614

    DISCOVERING NOVEL COMPOUNDS TO STABILIZE THE E2F-RETINOBLASTOMA PROTEIN COMPLEX IN THE CELL CYCLE

    Elise Brown, Cameron Pye, Tyler Liban, Seth Rubin.

    University of California, Santa Cruz, Santa Cruz, CA.

    The retinoblastoma protein (RB) is a key cell-cycle regulator. Mutations in the RB pathway cause rapid cell division, which is a hallmark of cancer. RB binds and inhibits the E2F transcription factor until cells are ready to divide. RB is then phosphorylated by cyclin-dependent kinases, which leads to E2F dissociation and its activation of cell-cycle genes. The structure of phosphorylated RB was previously determined, and when phosphorylated, its pocket domain and N-terminal domains associate to change the active site and release E2F. Many cancers have increased kinase levels which leads to phosphorylated RB, unbound E2F, and rapid cell division. We hypothesize that if a novel compound can be found to cause phosphorylated RB to have an affinity to E2F, a drug can be developed that stops rapid cell proliferation. Such a compound may block the site of pocket-N-terminal association or work by some other mechanism. We previously used a high-throughput fluorescence polarization (FP) anisotropy to identify lead compounds that increase E2F binding to phosphorylated RB. In current work, we have repeated the FP assay on screen hits prepared from fresh powders and followed up those results with isothermal titration calorimetry (ITC) experiments. The goal of ITC is to determine the affinity of the RB-E2F complex in the presence of the compound. To date, we have found 3 compounds that may show promise of enhancing binding of E2F to phosphorylated RB.

    THU-603 MUTATION OF AGOUTI SIGNALING PROTEIN FOR IMPROVING THE TREATMENT OF MELANOMA

    • Rodrigo Andrade ;
    • Glenn Millhauser ;

    THU-603

    MUTATION OF AGOUTI SIGNALING PROTEIN FOR IMPROVING THE TREATMENT OF MELANOMA

    Rodrigo Andrade, Glenn Millhauser.

    University of California, Santa Cruz, Santa Cruz, CA.

    Melanoma causes a large majority of skin cancer deaths. According to the American Cancer Society, approximately 10,000 people are expected to die of melanoma this year in the U.S. Melanoma is difficult to treat because melanosomes, organelles containing the pigment eumelanin, prevent chemotherapy from working. Agouti signaling protein (ASIP) binds to melanocortin receptor 1 and suppresses the production of eumelanin; thus, fewer melanosomes are formed and their ability to absorb and inactivate chemotherapeutics is minimized. When cells are treated with ASIP, chemotherapy has been shown to be 3 times more successful in treating melanoma. In order to continue using ASIP to treat melanoma, ASIP is synthesized using solid phase peptide synthesis, oxidative folding, and purification using HPLC. It will then be further tested for its effectiveness in melanoma treatment in model organisms.

    FRI-604 DESIGN OF SYNTHETIC PROTEIN SCAFFOLDING DRIVEN BY AN UNNATURAL AMINO ACID MEDIATED CONJUGATION REACTION

    • Sophia Tan ;
    • Nancy Hernandez ;
    • Sagar Khare ;

    FRI-604

    DESIGN OF SYNTHETIC PROTEIN SCAFFOLDING DRIVEN BY AN UNNATURAL AMINO ACID MEDIATED CONJUGATION REACTION

    Sophia Tan, Nancy Hernandez, Sagar Khare.

    Rutgers, The State University of New Jersey, New Brunswick, NJ.

    Synthetic protein scaffolding has the potential to optimize many biological processes and greatly improve reaction rates which lends to its appeal for use in industrial-scale applications such as organic synthesis of drugs, bioremediation, and biofuel production. By co-localizing pathway enzymes, these scaffolds are able to effectively decrease transit time of intermediates, minimize metabolite accumulation, protect unstable intermediates from solvent, and prevent escape of potentially toxic intermediates. However, previously constructed synthetic scaffolds have been limited by the natural binding-disassociation flux of pathway enzymes to the scaffolding complex. As a result, this project aims to computationally design and experimentally validate a set of PDZ domains that can be irreversibly linked to their high-affinity cognate peptides through a thiol-bromoethyl SN2 reaction between a bromine-containing unnatural amino acid (UAA) and a cysteine. In turn, pathway enzymes can be tethered onto the PDZ scaffolding complex through integration of the corresponding PDZ peptide. Rosetta was used for in silico modeling and design, and amber suppression with an engineered synthetase/tRNA pair was used to genetically incorporate the UAA in vitro. Following protein expression of the designed PDZ domains and their respective peptides, amber suppression will be measured, thioether bond formation will be assayed, and peptide binding will be characterized. Preliminary results include successful identification of positions to place the UAA and cysteine residues using the Rosetta matching algorithm. The structures were then subjected to RosettaDesign and ranked according to several specified parameters. This project is still in progress; complete results will be presented.

    FRI-614 RECONSTITUTION OF AN ARGONAUTE OPERON PUTATIVELY INVOLVED IN BACTERIAL IMMUNITY

    • Bridget Hansen ;
    • Kevin Doxzen ;
    • Jennifer Doudna ;

    FRI-614

    RECONSTITUTION OF AN ARGONAUTE OPERON PUTATIVELY INVOLVED IN BACTERIAL IMMUNITY

    Bridget Hansen1, Kevin Doxzen2, Jennifer Doudna2.

    1San Francisco State University, San Francisco, CA, 2University of California, Berkeley, Berkeley, CA.

    Argonaute proteins (Ago), the core proteins involved in RNA interference (RNAi), are essential in eukaryotic post-transcriptional gene regulation. Although the structure and function of eukaryotic Agos have been well studied, the role of their prokaryotic homologues remains elusive. Recent evidence suggests a role in prokaryotic immunity, but further investigation is required to verify these claims. A well-conserved operon composed of a restriction enzyme, helicase, nuclease, and Ago proteins has been identified in the organism Shewanella sp. ANA-3. We hypothesize this operon functions in phage defense rather than gene regulation. Demonstrated through RT-PCR, all 4 genes are transcribed together in a polycistronic transcript, suggesting a concerted function. We have overexpressed in E. coli and optimized the purification of all 4 proteins. We attempted reconstitution with the purified proteins to observe complex formation in vitro. Under experimental conditions, complex formation was not detected, suggesting that additional endogenous factors or stimuli must be present to initiate and stabilize protein interactions. Our next steps will focus on protein complex assembly in vivo. We will pull down endogenously tagged operonic proteins and identify any additional proteins or nucleic acid that remain associated. Observance of complex formation would provide a first hint at a putative prokaryotic defense system. Our biochemical and structural analysis of this novel clade of bacterial Agos will also lead to a more comprehensive understanding of argonaute evolution.

    FRI-622 THE EFFECTS OF REORIENTATION ON THE PHOTOSYNTHESIS OF POPLAR LEAVES

    • Michael Bennett ;
    • David Hanson ;

    FRI-622

    THE EFFECTS OF REORIENTATION ON THE PHOTOSYNTHESIS OF POPLAR LEAVES

    Michael Bennett, David Hanson.

    The University of New Mexico, Albuquerque, NM.

    The anatomy of a leaf works hand in hand with the biochemistry to maintain homeostasis within a leaf. While a mature leaf’s anatomy cannot be altered, it may be possible to alter its biochemistry and its photosystem function. This leads to the question: how can we perturb leaf biochemistry? Over the course of this summer, several tests were initiated involving reorientation of poplar leaves to identify correlated changes in the biochemical behavior and photosynthetic function. Mature poplar leaves were flipped upside down and pinned so that incident light would enter the bottom of the leaf rather than the top. The leaf's photosynthetic function and biochemistry were monitored using several different tests, including a Li-cor machine, which tests gas exchange within the leaf; chlorophyll assay, which calculates the amount of chlorophylls a and b in a fresh leaf sample; and PAM (pulse amplified modulation) fluorescence, which calculates the electron transport rate by testing both effective and optimal quantum yield. The reoriented leaf was compared to 2 control orientations: one was left in its natural orientation, which was slightly vertical; the other was pinned completely horizontal so it would remain constantly in the sunlight. Leaf reorientation was found to result in impaired photosystem function, specifically a reduction in optimal quantum yield; therefore, reorientation did affect the biochemistry of the leaf.

    FRI-620 SYNTHESIS AND USE OF BIOTINYLATED NAADPS FOR THE AFFINITY PURIFICATION OF THE NAADP RECEPTOR

    • Natalia Olmeda ;
    • Timothy Walseth ;

    FRI-620

    SYNTHESIS AND USE OF BIOTINYLATED NAADPS FOR THE AFFINITY PURIFICATION OF THE NAADP RECEPTOR

    Natalia Olmeda1, Timothy Walseth2.

    1University of Puerto Rico in Humacao, Humacao, PR, 2University of Minnesota, Minneapolis, MN.

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger molecule that activates calcium release from intracellular acidic stores such as lysosomes. Calcium release mediated by NAADP can result in both local and global signaling cascades within a cell, triggering other calcium signaling pathways. Calcium signaling is important in cellular functions such as fertilization, muscle contraction, and gene expression. Two-pore channels are a family of ion channels that have been identified in the NAADP pathway; however, the receptor protein for NAADP has not been identified. The purpose of this study is to synthesize biotinylated NAADPs for purification of the NAADP receptor by affinity chromatography. Four biotinylated NAADPs were successfully synthesized and characterized for their ability to mimic NAADP action in terms of binding and calcium release in the sea urchin system. All 4 biotinylated NAADPs were able to successfully compete with 32P-NAADP in a competition binding assay with IC50 values 10- to 1000-fold higher than NAADP itself. These compounds also were able to release calcium with EC50 values 125- to 1200-fold higher than NAADP. These probes desensitized calcium release induced by 1µM NAADP with IC50 values similar to those observed in the competition binding assays. Immobilization of the biotinylated NAADPs on streptavidin-agarose for affinity purification of NAADP binding proteins resulted in the retention of several proteins compared to control columns. This approach might prove useful in the identification of the NAADP receptor.

    FRI-607 DEVELOPING A CONTINUOUS COLORIMETRIC KINETIC ASSAY TO MEASURE HUMAN CYSTATHIONINE-SYNTHASE (CBS) ACTIVITY

    • Dana Kennedy ;
    • Raymond Esquerra ;
    • Bushra Bibi ;
    • George Gassner ;

    FRI-607

    DEVELOPING A CONTINUOUS COLORIMETRIC KINETIC ASSAY TO MEASURE HUMAN CYSTATHIONINE-SYNTHASE (CBS) ACTIVITY

    Dana Kennedy, Raymond Esquerra, Bushra Bibi, George Gassner.

    San Francisco State University, San Francisco, CA.

    Human cystathionine β-synthase (hCBS) and cystathionine γ-lyase (hCSE) are 2 key enzymes in the transsulfuration pathway and are essential in the production of sulfur containing amino acids. CBS catalyzes the conversion of homocysteine and serine into cystathionine, which is later converted to cysteine by hCSE. Naturally occurring mutations reduce the ability of hCBS to remove homocysteine resulting in hyperhomocysteinemia, a disorder associated with a greater risk for developing cardiovascular and neurological diseases. Current assays for hCBS activity are fixed-time measurements that involve 5,5'-dithiobis-(2-nitrobenzoic acid), radiolabeling, and high performance liquid chromatography. The goal of this project is to develop a simple, continuous, kinetic assay to monitor CBS activity. We tested 3 methods: a coupled kinetic assay with hCSE and lactate dehydrogenase to determine hCBS production of cystathionine by measuring the oxidation of NADH to NAD+; a coupled kinetic assay in which cystathionine produced from CBS undergoes oxidative deamination by L-amino acid oxidase to yield alpha- keto acid, which is detected with ninhydrin reagent; and a coupled kinetic assay to hCSE in which cystathionine is monitored from its rapid conversion into cysteine using a fluorophore selective to cysteine. To test these methods, we determine the kinetics of wildtype and a truncated CBS mutant lacking the regulatory domain. Preliminary results show that these assay methods seem likely to be simple and accurate methods to continuously monitor CBS kinetic activity. The long term goal of this project is to better understand how the heme domain modulates and regulates the activity of hCBS.

    THU-621 NEW OPPORTUNITIES IN BIOAVAILABILITY OPEN UP FOR CYCLIC PEPTIDES

    • Jose Garcia ;
    • Christain Etienne ;
    • Rushia Turner ;
    • Scott Lokey ;

    THU-621

    NEW OPPORTUNITIES IN BIOAVAILABILITY OPEN UP FOR CYCLIC PEPTIDES

    Jose Garcia, Christain Etienne, Rushia Turner, Scott Lokey.

    University of California, Santa Cruz, Santa Cruz, CA.

    Peptides have many key roles in biology, from transmitting signals in the brain to binding to receptors on cells. N-methylated cyclic peptides have gained attention in drug discovery because they are more stable to proteolysis, and they are more likely to exhibit passive membrane permeability. The Lokey Research Group has exhaustively examined the pharmacokinetics of the multiple N-methylated cyclic peptide cyclo[L-Leu-L-Leu-NMe-D-Leu-NMe-L-Leu-L-Tyr-NMe-D-Pro], also known as 1NMe3. This compound was shown to have oral bioavailability of 28% in rats, but it was extensively metabolized in the liver via N-demethylation. Three analogs of 1NMe3 are being synthesized in which the ­­N-methylated leucine residues are replaced with N-isobutyl alanine residues, both individually and in tandem. These analogs represent the starting point for a larger investigation into the effect of bulky N-alkyl substituents on the membrane permeability and hepatic stability of cyclic peptides. The passive membrane permeability of the three N-isobutyl alanine-containing compounds will be tested using both cell-free and cell-based assays. Hepatic stability will be evaluated using an in vitro human liver microsome assay. The membrane permeability and hepatic stability data will then be used to develop and synthesize more extensive N-alkylated cyclic peptide libraries.

    FRI-606 OVER EXPRESSION OF THE ALDEHYDE DEHYDROGENASE GENE FROM HALOPIGER XANADUENSIS IN HALOFERAX VOLCANII TO PRODUCE BUTANOL

    • Jazel Hernandez ;
    • David Bernick ;

    FRI-606

    OVER EXPRESSION OF THE ALDEHYDE DEHYDROGENASE GENE FROM HALOPIGER XANADUENSIS IN HALOFERAX VOLCANII TO PRODUCE BUTANOL

    Jazel Hernandez, David Bernick.

    University of California, Santa Cruz, Santa Cruz, CA.

    The University of California Santa Cruz International Genetically Engineered Machine (iGEM) team is engineering Haloferax volcanii to produce the solar-based liquid biofuel, butanol, from glucose. In 2014, the team investigated gene knockouts in fatty-acid synthesis Acyl- CoA (ACD) 2,3, and 4, as a means to accumulate butyryl-CoA. These knockouts prevented growth on glucose, however. This year the focus is to find genes that can be overexpressed in this organism to produce butanol. The 2 genes believed to be between butyryl-CoA and butanol are aldehyde dehydrogenase and alcohol dehydrogenase. The aldehyde dehydrogenase (alDH) gene chosen is from Halopiger xanadensis, a close relative of H. volcanii; they are both salt-loving extremophiles. This alDH gene is inserted using gibson assembly into our expression plasmid (pTA963) which then is transformed into H. volcanii. We are expecting that the alcohol gene (ADH2), which is already in Haloferax volcanii, turns on as a result of acidified media that the organism is growing in. We hypothesize that by acidifying the growth media and inserting the aldehyde gene into H. volcanii, butanol will then be produced. In order to quantify the butanol production by both the genetically engineered cultures and the wild-type, gas chromatography will be used.

    FRI-600 LARVAL AGE DETERMINATION IN FORENSICALLY IMPORTANT INSECT SPECIES USING BIOCHEMICAL AND FLUORESCENCE MICROSCOPY TECHNIQUES

    • Caitlyn Tobita ;
    • Helen Turner ;
    • Danielle Flores ;
    • Amy Miller-Weber ;
    • Angelique Showman ;
    • Lori Shimoda ;
    • Carl Sung ;
    • David Carter ;

    FRI-600

    LARVAL AGE DETERMINATION IN FORENSICALLY IMPORTANT INSECT SPECIES USING BIOCHEMICAL AND FLUORESCENCE MICROSCOPY TECHNIQUES

    Caitlyn Tobita, Helen Turner, Danielle Flores, Amy Miller-Weber, Angelique Showman, Lori Shimoda, Carl Sung, David Carter.

    Chaminade University of Honolulu, Honolulu, HI.

    Entomological methodologies for analyzing fly larvae for estimating the time of colonization (TOC) are commonly used to assist in death investigations. While the methodologies for analyzing fly larvae differ, most rely on light microscopy or electron microscopy. The former is low resolution but tractable, the latter is resource intensive and suitable for only small sample sets. This study served as a proof of concept in the forensically important Chrysomya rufifacies, where we sought to combine high-content fluorescence miscopy and biochemical measures of developmental marker proteins to improve resolution of larval age. We applied confocal microscopy and observed robust, non-bleaching, autofluorescence of appropriately prepared larval posterior sections. Here we describe a pilot study that established fixation and mounting protocols, defined a set of measurable morphometric criteria, and captured developmental transitions of second instar to third instars using both fluorescence microscopy and anti-ecdysone receptor western blot analysis. The data show that these instar stages can be distinguished on the basis of epi-fluorescent microscopy and confocal microscopy. High content imaging techniques using confocal microscopy, combined with morphometry and biochemical techniques, may therefore aid forensic entomologists in determining estimated TOC.

    THU-622 ALTERED PYRUVATE KINASE MRNA EXPRESSION AND ABNORMAL METABOLIC PROFILES IN MELANOMA CELLS EXPRESSING THE WARBURG EFFECT

    • Jonathan McKinney ;
    • Todd Thompson ;

    THU-622

    ALTERED PYRUVATE KINASE MRNA EXPRESSION AND ABNORMAL METABOLIC PROFILES IN MELANOMA CELLS EXPRESSING THE WARBURG EFFECT

    Jonathan McKinney1, Todd Thompson2.

    1McDaniel College, Westminster, MD, 2The University of New Mexico, Albuquerque, NM.

    Many cancer cells preferentially undergo lactic acid fermentation from glycolysis rather than oxidative phosphorylation. Even with adequate amounts of oxygen to metabolize glucose that could yield higher amounts of energy, cancer cells produce lactic acid in a process called the Warburg effect. Moreover, melanoma cancers can exhibit excessive lactic acid production, as was found in the SKMEL19 and SKMEL29 melanoma cell lines that exhibit the Warburg effect. Phenotypic changes that may facilitate the Warburg effect include alterations in the expression of splice variants of pyruvate kinase (PK), which performs the final step in glycolysis, and increased expression of lactate dehydrogenase A (LDH-A), the enzyme responsible for producing lactic acid from pyruvate. Surprisingly, in the current study, gene expression measured using QPCR showed decreased levels of LDH-A mRNA in SKMEL19 and SKMEL29 melanoma cells compared to cancer cells that do not undergo this process (i.e., PC3 and SKMEL103 cells). However, increased LDH protein expression was observed in these cells. Inhibition of LDH via oxamic acid led to decreased growth. Levels of PK mRNA isoforms PKM1 and PKM2 were also determined, the latter being characteristic of cancers exhibiting the Warburg effect. The ratio of PKM2 to PKM1 mRNA was found to be increased in SKMEL19 and SKMEL29 cells, ultimately suggesting PKM2 as a key player in mediating the Warburg effect. Our results suggest that cancer cells exhibiting the Warburg effect have developed highly coordinated metabolic adaptations that promote cancer growth.

    THU-606 ISOLATION AND CHARACTERIZATION OF A MEMBRANE-BOUND PINK PIGMENT FROM ACIDOBACTERIUM CAPSULATUM

    • Elena Mylroie ;
    • Tara Abrams ;
    • Leslie Sommerville ;

    THU-606

    ISOLATION AND CHARACTERIZATION OF A MEMBRANE-BOUND PINK PIGMENT FROM ACIDOBACTERIUM CAPSULATUM

    Elena Mylroie, Tara Abrams, Leslie Sommerville.

    Fort Lewis College, Durango, CO.

    Acidobacterium capsulatum was cultured from acid mine drainage, has optimal growth at pH 3.0 to 6.0, is a chemoorganotroph, contains membrane menaquinones, and its genotypes have been identified in environments worldwide. When these organisms are grown in liquid media with glucose as the sole carbon source under aerobic conditions the cells become pink, allowing the culture to take on the shade of pink lemonade as the cells transition from log to stationary phase. However, when these cells are grown under the same conditions with septum-sealed vials the cells remain white. The key question being asked in this study is: what is the best method to extract and characterize this pink pigment? Extraction methods involving cell lysis or not, using methanol, isopropanol, diethyl ether, and various combinations were tried. It was found that a methanol/diethyl ether-based extraction was the best with the key membrane components extracted in the diethyl ether phase. This phase contained 3 membrane pigments with characteristic UV-VIS absorbance spectra and unique elution times on a C-18 reverse phase HPLC column eluted with a linear gradient of 1% phosphoric acid to 100% acetonitrile. Final identification of each pigment will be made based on comparison of UV-VIS spectra, elution times, and mass spectra analysis relative to standard compounds. This analysis will be done with both pink and white cells. The preliminary structures of each of these pigments will be completed this fall. Future studies will look at the distribution and function of the pink pigment in A. capsulatum.

    THU-602 TRANSACTIVATION DOMAIN BINDING OF E2F TRANSCRIPTION FACTORS

    • Miguel Osorio Garcia ;
    • Caileen Brison ;
    • Tyler Liban ;
    • Seth Rubin ;

    THU-602

    TRANSACTIVATION DOMAIN BINDING OF E2F TRANSCRIPTION FACTORS

    Miguel Osorio Garcia, Caileen Brison, Tyler Liban, Seth Rubin.

    University of California, Santa Cruz, Santa Cruz, CA.

    Regulation of cell transition into the DNA replication stage is a keystone process of the cell cycle. The E2F family of transcription factors has been shown to control the expression of genes which facilitate this transition and other cell cycle control functions. E2Fs are in turn regulated by Rb through inhibitory binding. This binding blocks the E2F transactivation domain, impeding transcription. Rb is phosphorylated before cell cycle initiation, which leads to its dissociation and in turn allows E2F regulated genes to be transcribed. This process has been widely studied as cell cycle deregulation is tightly associated with uncontrolled cell division and cancer. Although E2Fs have been proven to be such important regulators, direct study of their interactions with complexes besides Rb is lacking. We aim to understand the mechanism of gene activation by E2Fs and hypothesize that the transactivation domain binds proteins that stimulate transcription such as RNA polymerase and CBP-KIX. We intend to investigate these interactions through biophysical methods such as isothermal titration calorimetry and nuclear magnetic resonance. These techniques, which detect changes in the physical and chemical environment of proteins upon association, will provide evidence for specific interactions in vitro and will allow for structural characterization of E2F-activator complexes. Of the combinations tested thus far, no binding has been observed, but this could be due to measurement limitations. This investigation will lead to a better understanding of cell division regulation and other methods of E2F control, allowing the continuation of the study of cancer and its deregulation effects.

    FRI-616 PRODUCTION OF 2-ISOPROPYLMALATE THROUGH BIOENGINEERED E. COLI: A BIO-BASED PATHWAY TOWARD CONJUGATED DIENES

    • Susan Pham ;
    • Jingyu Wang ;
    • Kechun Zhang ;

    FRI-616

    PRODUCTION OF 2-ISOPROPYLMALATE THROUGH BIOENGINEERED E. COLI: A BIO-BASED PATHWAY TOWARD CONJUGATED DIENES

    Susan Pham1, Jingyu Wang2, Kechun Zhang2.

    1Oklahoma State University, Stillwater, OK, 2University of Minnesota, Minneapolis, MN.

    Thirty million metric tons of rubbers are produced yearly worldwide; two-thirds of this amount comes from nonrenewable, petroleum-derived sources. Because of the growing market and the decreasing supply of petroleum and crude oil sources, there comes a need for a sustainable avenue for production. The companies Dupont and Goodyear have produced isoprene, which is a monomer for natural rubber, through a sustainable bioprocess. However, only 1 mole of isoprene is produced per 1.5 mole glucose. Isoprene is also very volatile, which affects the process’s productivity and can cause environmental concerns. To optimize the efficiency and counteract the problem, we have developed a bio-based route to produce 2-isopropylmalate, a precursor for an isoprene analog. The analog is proposed to show similar qualities to isoprene while being less volatile, thus having a lower impact on the environment. In this work, we designed a biosynthetic route to produce 2-isopropylmalate from glucose in non-pathogenic Escherichia coli. The genes ilvD, leuA, and alsS were cloned into a pZE vector and transformed into the E. coli strain BW25113. The transformants were incubated overnight at 37 °C. Afterwards, the transformed bacteria was inoculated into a fermentation medium containing 40 g/L glucose; this was put in a 30 °C shaker for 48 hours. Through analysis of high performance liquid chromatography data, the titer of 2-isopropylmalate was 8.04 g/L and the product yield was 29.1% of the theoretical yield.

    THU-608 BIOSYNTHESIS OF SILVER NANOPARTICLES USING LACTOBACILLUS ACIDOPHILUS

    • Itzel Tejeda ;
    • Karina Castillo ;

    THU-608

    BIOSYNTHESIS OF SILVER NANOPARTICLES USING LACTOBACILLUS ACIDOPHILUS

    Itzel Tejeda, Karina Castillo.

    El Paso Community College, El Paso, TX.

    A green approach in the fabrication of silver nanoparticles is of considerable importance. It can have a positive impact on the environment, expand their biomedical applications, and benefit the market by making nanoparticles less costly. Therefore, it is important for new methods to be developed. Current literature has not identified in detail the effects of culture conditions on nanoparticle characteristics, specifically size and morphology, when using Lactobacillus acidophilus. This research presents an eco-friendly method for biosynthesis of silver nanoparticles using Lactobacillus acidophilus as the reducing agent and on silver nitrate (AgNO3) as the source of the metal. The synthesis of nanoparticles will be studied under 5 conditions: pH, temperature, substrate concentration, time of reaction, and bacteria to metal ratio. It has been reported that smaller silver nanoparticles have an absorption peak in the 400-500 nm range; thus, we predict that a basic solution, temperature greater than 50 °C, 1 mM AgNO3, a concentration ratio of Lactobacillus acidophilus to AgNO3 of 1:2, and a time of reaction upward of 60 minutes will optimize the production of smaller, mono-dispersed particles with a spherical shape. Preliminary results from ultraviolet-visible spectrophotometry showed an absorbance peak at the 400 nm range with an optimal pH of 11 and AgNO3 concentration of 1 mM suggesting a size of 20 nm. Results for temperature, time of reaction, and bacteria to metal ratio are still pending. For characterization of nanoparticle morphology and confirmation of size and dispersion pattern in solution, transmission electron microscopy, and X-ray diffraction techniques will be used.

    FRI-618 ASSEMBLY OF MITOCHONDRIAL SUPERCOMPLEXES IN THE YEAST SACCHAROMYCES CEREVISIAE

    • Zoe Cosner ;
    • Flavia Fontanesi ;
    • Antoni Barrientos ;

    FRI-618

    ASSEMBLY OF MITOCHONDRIAL SUPERCOMPLEXES IN THE YEAST SACCHAROMYCES CEREVISIAE

    Zoe Cosner, Flavia Fontanesi, Antoni Barrientos.

    University of Miami, Miami, FL.

    The mitochondrial oxidative phosphorylation system, composed of mitochondrial respiratory chain (MRC) complexes I-IV, ATP synthase, and two electron carriers, provides most cellular energy in the form of ATP. MRC complexes form ordered structures called supercomplexes, which have been proposed to enhance individual complex stability and increase respiratory efficiency. Supercomplex organization is well characterized in lower to higher eukaryotes. However, despite their importance, supercomplex assembly remains poorly understood. Several observations in yeast and mammalian cells have suggested that structural subunits of different complexes may interact in subassembly intermediates. We hypothesize that supercomplexes do not originate from the association of preassembled individual complexes. Rather, their biogenesis involves the incorporation of partially assembled complexes and free subunits into subassembly intermediates of increasing complexity. To investigate the molecular mechanisms involved in supercomplex assembly, we use the yeast Saccharomyces cerevisiae as a model system. Yeast supercomplexes are composed of a complex III dimer and 1 or 2 complex IV monomers. We have engineered a system that allows for regulatable expression of CIII and CIV subunits. Our novel yeast models allow us to halt a specific MRC subunit's expression and restore it at any moment after the MRC enzymes have been turned over, thus facilitating the study of MRC supercomplex assembly pathways de novo. Our initial data show that some CIII and CIV subunits are incorporated directly into supercomplexes in late assembly stages, while others accumulate in earlier subassemblies, which could represent supercomplex assembly intermediates. We are now in the process of further characterizing the step-by-step supercomplex assembly pathway.

    FRI-619 CONSTRUCTION OF RNA BINDING PROTEIN (RBP) EXPRESSION VECTORS, RPBS PURIFICATION, AND ANTIBODY PRODUCTION TO FURTHER UNDERSTAND THE ROLE OF RBP COMPLEXES IN RICE STORAGE PROTEIN MRNAS TRANSPORT

    • Johanna Bautista ;
    • Hong-li Chou ;
    • Thomas Okita ;

    FRI-619

    CONSTRUCTION OF RNA BINDING PROTEIN (RBP) EXPRESSION VECTORS, RPBS PURIFICATION, AND ANTIBODY PRODUCTION TO FURTHER UNDERSTAND THE ROLE OF RBP COMPLEXES IN RICE STORAGE PROTEIN MRNAS TRANSPORT

    Johanna Bautista1, Hong-li Chou2, Thomas Okita2.

    1California State University, Los Angeles, Los Angeles, CA, 2Institute of Biological Chemistry, Washington State University, Pullman, WA.

    Key functions of seed storage proteins are to serve as a nitrogen reserve that supports growth of the germinating seedling. These storage proteins also serve as a major source of dietary proteins for much of the world’s population. Rice accumulates 2 major classes of storage proteins, prolamines and glutelins, which accumulate in different subcellular locations; the ER-lumen to form protein body-I (PB-I) and the protein storage vacuole (PSV, PB-II), respectively. Earlier studies have demonstrated that RNA localization to the cortical ER is not random. Prolamine RNAs are targeted to the PB-ER that bound the PB-I, while glutelin RNAs are localized to adjacent interconnecting cisternal ER. Subsequent studies show that storage protein RNA localization is a multi-step processes requiring several cis-elements located in the coding sequences and 3’UTRs of the prolamine and glutelin RNAs. We have identified several potential RBPs that may serve as potential RNA localization trans-factors that bind to the cis-elements of the storage protein mRNAs. To generate antibodies to these RNA binding proteins (RBPs), a vector was constructed that facilitates efficient RBP cloning and expression as well as subsequent purification of the expressed protein by immobilized metal-affinity chromatography. Using this expression plasmid, results describing the optimized expression of 3 recombinant RBPs that interact with OsTudor-SN, a key RBP required for storage protein RNA localization, will be presented. Our studies will advance the understanding of how RNA transport complexes are formed and remodeled during the movement of mRNAs to specific cellular locations in rice endosperm tissue.

    FRI-603 INCREASING THE BINDING AFFINITY OF ASIP TO MC1R BY MODIFICATION OF THE LIGAND'S C-TERMINAL LOOP

    • Cynthia Lai ;
    • Glenn Millhauser ;

    FRI-603

    INCREASING THE BINDING AFFINITY OF ASIP TO MC1R BY MODIFICATION OF THE LIGAND'S C-TERMINAL LOOP

    Cynthia Lai, Glenn Millhauser.

    University of California, Santa Cruz, Santa Cruz, CA.

    The melanocortin system regulates pigmentation, UV protection, inflammation, and energy balance. The melanocortin-1 receptor (MC1R) specifically has a role in pigmentation and UV protection. The disulfide-rich antagonist agouti signaling protein (ASIP) binds to MC1R resulting in the production of pheomelanin. On the other hand, the agonist α-melanocyte-stimulating hormone (α-MSH) results in the production of eumelanin. Melanocytes naturally produce eumelanosomes, which are small granules containing eumelanin that protects DNA from UV radiation. In melanoma, chemotherapy is ineffective because eumelanosomes export anticancer drugs out of the cell. By modifying ASIP, we can enhance its binding affinity to MC1R and decrease the production of eumelanosomes. We focused on varying the sequence of the C-terminal loop of ASIP because the C-terminal loop is necessary for inverse agonist activity specifically at MC1R. We replaced serine, the 129th amino acid of wild-type ASIP, with leucine to improve hydrophobic interactions with the receptor. Using a CEM liberty microwave peptide synthesizer, mutant ASIP S129L was prepared on an H-rink amide resin and then cleaved under acidic conditions. The peptide was purified with reverse-phase high performance liquid chromatography (HPLC) and electrospray mass spectrometry. We have purified pure, unfolded ASIP S129L and are currently optimizing the oxidative fold conditions. We plan to vary the solvent, adjust the levels of oxidizing agents, and change the amount of disulfide shuffling enhancers. Overall, folded ASIP S129L is predicted to have stronger hydrophobic interactions with MC1R and could become a helpful therapeutic for skin cancer chemotherapy.

    FRI-611 UNDERSTANDING HOW THE DISTAL POCKET ENVIRONMENT AFFECTS THE BINDING OF NITRITE TO HEME PROTEINS

    • Adriana Garcia ;
    • Rocio Gomez ;
    • Jee Kim ;
    • Raymond Esquerra ;

    FRI-611

    UNDERSTANDING HOW THE DISTAL POCKET ENVIRONMENT AFFECTS THE BINDING OF NITRITE TO HEME PROTEINS

    Adriana Garcia, Rocio Gomez, Jee Kim, Raymond Esquerra.

    San Francisco State University, San Francisco, CA.

    Nitric oxide supports a variety of physiological processes, and proper nitric oxide metabolism is essential for cardiovascular health. Heme proteins can support vasodilation during hypoxia by converting nitrite (NO2-) to nitric oxide (NO); however, the factors that govern how effective a heme protein is at this chemistry are poorly understood. Our research aims to understand how the distal pocket environment affects the binding affinity and binding mode of NO2- to met-myoglobin using spectrophotometry and magnetic circular dichroism (MCD) spectroscopy. We compare the binding affinity of distal pocket mutants that affect hydrogen bonding and size of the distal pocket and correlate coordination chemistry with binding affinity to establish a clear picture of how the protein environment controls nitrite binding in heme proteins. We demonstrate that the removal of the distal hydrogen bond increases the N-Nitro mode of binding and decreases NO2- binding affinity significantly. Nonpolar distal residues reduce the NO2- binding affinity and the O-Nitrito mode of binding in the following order: H64A < H64L < H64V. Both H64Q and V66F bind NO2- tighter than WT, due to a stronger hydrogen bond between the nitrite and the distal histidine that favors the O-Nitrito confirmation. Reducing the distal pocket using L29 mutants reduces O-Nitrito binding by the same factor while L29F < L29W for NO2- binding affinity. We demonstrate that NO2- binding affinity and binding mode is controlled via electrostatic and steric interactions between NO2- and the protein matrix. Understanding how the protein environment influences nitrite binding in heme proteins helps toward understanding how these proteins generate NO physiologically.

    FRI-613 SURVIVING WITH HYDROGEN SULFIDE: UNDERSTANDING HOW PROTEINS CONTROL SULFHEME FORMATION

    • Pedro Tuyub ;
    • Raymond Esquerra ;
    • Emily Zepeda ;

    FRI-613

    SURVIVING WITH HYDROGEN SULFIDE: UNDERSTANDING HOW PROTEINS CONTROL SULFHEME FORMATION

    Pedro Tuyub1, Raymond Esquerra1, Emily Zepeda2.

    1San Francisco State University, San Francisco, CA, 2City College of San Francisco, San Francisco, CA.

    Hydrogen sulfide in the past was only known for its toxicity; however, it was recently discovered to be a gasotransmitter at low concentrations in the mammalian body. Studies have shown that hydrogen sulfide interacts with heme proteins by modifying the pyrrole ring through sulfheme formation. Heme proteins play a role in hydrogen sulfide degradation, and are essential to the regulation of H2S concentrations in the body. Because different heme proteins vary in their susceptibility to sulfheme formation, this work systematically explores how the protein matrix influences the formation of sulfheme. Specifically, we use wild-type and mutant sperm whale myoglobins to examine how the distal pocket environment affects the kinetics of sulfheme formation. We hypothesize that making the distal pocket less polar and reducing pocket volume will decrease the rate of sulfheme formation. Wild-type and mutant myoglobins H64A, H64L, H64V, H64Q, L29F, L29W, and V68F were exposed to hydrogen sulfide under different environmental conditions. These environmental factors include oxygen concentration, pH, and temperature, which are known to influence sulfheme production. We measured sulfheme formation kinetics spectrophotometrically. Defining how the distal pocket controls sulfheme formation is essential toward understanding how hydrogen sulfide is managed and produced physiologically.

    THU-616 DISSECTING TERTIARY LYMPHOID TISSUES IN PANCREATIC ISLETS OF AUTOIMMUNE DIABETES

    • Fatimata Sanogo ;
    • Wenxian Fu ;
    • Asami Yamamura ;
    • Yi Dong ;
    • Xiaomei Yuan ;

    THU-616

    DISSECTING TERTIARY LYMPHOID TISSUES IN PANCREATIC ISLETS OF AUTOIMMUNE DIABETES

    Fatimata Sanogo, Wenxian Fu, Asami Yamamura, Yi Dong, Xiaomei Yuan.

    University of California, San Diego, La Jolla, CA.

    Type I diabetes (T1D) is an organ-specific autoimmune disease. This disorder is the result of the destruction of beta cells (the only insulin-producing cells in the body) by the immune system. It is a critical health and social problem because the incidence of T1D has dramatically increased in the last few decades, especially among children under the age of 5. The etiology of T1D is still unknown, but it is understood today that immune cell dysfunctions and autoimmunity play critical roles in the disease progression. In this project, we are interested in dissecting the various aspects of immune cell infiltration and organization in disease lesions in T1D. Using major animal models of T1D, we will comprehensively dissect the patterns of infiltrated immune cells into pancreatic islets at various stages of T1D unfolding and progression. We expect to define how the variety of immune cells assemble secondary lymphoid structures within the inflamed pancreatic islets and how these immune cells change their compositions over time during the disease development. These data will provide important clues for understanding the pathogenesis of this complex disease and help develop more targeted immune regulatory strategies for disease suppression.

    THU-601 SYNTHETIC STUDIES OF COLLETOIC ACID, A SELECTIVE 11-BETA-HYDROXYSTEROID DEHYDROGENASE INHIBITOR

    • Adam Putnam ;
    • Taotao Ling ;
    • Fatima Rivas ;

    THU-601

    SYNTHETIC STUDIES OF COLLETOIC ACID, A SELECTIVE 11-BETA-HYDROXYSTEROID DEHYDROGENASE INHIBITOR

    Adam Putnam, Taotao Ling, Fatima Rivas.

    St. Jude Children's Research Hospital, Memphis, TN.

    Colletoic acid is a novel acorane sesquiterpenoid isolated from the fungus Colletotrichum gloeosporioides, and exerts remarkable selective inhibition against 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). The 11β-HSD isoforms maintain homeostasis in the cell by regulating the conversion of cortisone to cortisol which is involved in various biological processes such as metabolism and cell growth. This biological profile renders colletoic acid, a potential molecular probe, to study important mechanisms mediated by 11β-HSD1 action. An efficient synthetic route to (+)-colletoic acid analogs has been developed in our laboratory. We hypothesized that a diversity-oriented strategy featuring a modified palladium mediated Heck reaction could provide the all-carbon spirocenter at the multigram scale to access other acorane family members and synthetic analogs. Compounds were purified and characterized using column chromatography, HPLC-MS, and NMR techniques. Our convergent synthetic strategy afforded gram quantities of the fully functionalized colletoic acid core, which can be used to develop other acorane family members. Our preliminary structure activity relationship (SAR) studies highlighting analogs synthesized via alpha-hydroxylation, chemoselective epoxidation, and halogenation will be disclosed.

    FRI-609 CHEMICAL PROFILING OF A MARINE-DERIVED, GRAM-NEGATIVE BACTERIA: ACHROMOBACTER SPANIUS

    • Alexis Munoz ;
    • Christine Theodore ;
    • Patrick Still ;
    • Phil Crews ;

    FRI-609

    CHEMICAL PROFILING OF A MARINE-DERIVED, GRAM-NEGATIVE BACTERIA: ACHROMOBACTER SPANIUS

    Alexis Munoz, Christine Theodore, Patrick Still, Phil Crews.

    University of California, Santa Cruz, Santa Cruz, CA.

    Secondary metabolites, unlike primary metabolites, are not involved in growth, development, or reproduction of an organism and often play a role in chemical defense and communication. Within the marine environment, secondary metabolites from non-photosynthetic Gram-negative bacteria are considered to be a promising source of new natural products. A bacterial strain that was isolated from a backshore (dry sand) sample collected at Zmudowski State Beach in California was identified as Achromobacter spanius: 100% identical to A. spanius by partial 16S rRNA gene sequence. The ethyl acetate crude extract of a liquid culture of this strain was subjected to pre-fractionation by high performance liquid chromatography (HPLC). The pre-fractions were analyzed using ultra high performance liquid chromatography (UPLC) and high-resolution accurate mass spectroscopy. The masses, accurate to 4 decimal places, obtained for each peak (A-R) were used to generate molecular formulas. These molecular formulas were then used to search compound databases such as Dictionary of Natural Products (DNP) and ChemSpider, with the goal of prioritizing the isolation of potentially new natural products. On examination of the mass spectra data obtained by the Orbitrap for peaks A-R, peak J proved to have an interesting mass due to its few matches in databases DNP and Chemspider. Further examination of peak J revealed 2 potential molecular formulas through the dereplication process of F-4 fraction, indicating a potential structure (C14H33N4O4S) sulfaquinoxaline.

    FRI-631 CONSTRUCTING ORTHOGONALITY BETWEEN PROTEIN DEGRADATION PATHWAYS

    • Nathan Rogers ;
    • William Mather ;

    FRI-631

    CONSTRUCTING ORTHOGONALITY BETWEEN PROTEIN DEGRADATION PATHWAYS

    Nathan Rogers, William Mather.

    Virginia Polytechnic Institute and State University, Blacksburg, VA.

    Proteins containing specific amino acid sequences known as degradation tags can be selectively degraded via corresponding proteases in the cell. The ClpXP protease in E. coli rapidly degrades proteins with the LAA tag, and this feature is often used in synthetic biology to build complex circuits such as gene oscillators. However, reliance on ClpXP alone limits the variety of synthetic circuits that can be constructed. Different ClpXP substrates have been shown to compete for common ClpXP machinery and thus interfere with each other. Synthetic oscillators that only share the common machinery of ClpXP have been shown to couple in an E. coli cell. No orthogonal synthetic oscillators have been developed, and all current bacterial synthetic oscillators rely on the ClpXP machinery. To increase the scalability of synthetic gene circuits, we are developing a variety of degradation tags that selectively target proteins to either the ClpXP or ClpAP protease in E. coli. We test orthogonality of these tags using precisely controlled synthetic gene circuits, measured using fluorescence spectrometry and microscopy. Our results are integrated using a queueing-theoretic formalism which leads to intuitive and quantitative predictions for our systems.

    THU-629 OXAMNIQUINE-DERIVATIVE DRUG TESTING IN ADULT MALE SCHISTOSOMES

    • Bethany Cruz ;
    • Philip LoVerde ;
    • Stacey Stahl ;
    • P. John Hart ;
    • Alexander Taylor ;

    THU-629

    OXAMNIQUINE-DERIVATIVE DRUG TESTING IN ADULT MALE SCHISTOSOMES

    Bethany Cruz2, Philip LoVerde1, Stacey Stahl1, P. John Hart1, Alexander Taylor1.

    1The University of Texas Health Science Center at San Antonio, San Antonio, TX, 2Texas Biomedical Research Institute, San Antonio, TX.

    Human schistosomiasis is a disease caused by blood-dwelling fluke worms of the genus Schistosoma. Schistosomiasis has been recognized as one of the 6 most neglected tropical diseases by the World Health Organization, and affects 200 million individuals globally. Most cases of human schistosomiasis are caused by the S. mansoni, S. haematobium, or S. japonicum species. Praziquantel (PZQ) is currently the only method of treatment available for the disease. As the drug of choice, PZQ is highly efficacious, but as a mono-therapy, the prospect of drug resistance remains a serious concern. Therefore, a second drug to be used in conjunction with PZQ is drastically needed at this time. The focus of this research is on oxamniquine (OXA), a species-specific prodrug that is enzymatically activated in S. mansoni and no other schistosome species. Through genetic analysis and RNA interference, a sulfotransferase (smSULT) was identified as responsible for the mechanism of action. The purpose of this research is to identify an OXA derivative that will kill S. haematobium. We screened OXA derivatives and the most efficacious one was tested against S. haematobium. Six drugs were tested in vitro, and one killed 40% of S. haematobium adult male worms. While drug experimentation is currently ongoing, this data is significant because it demonstrates that OXA derivatives can be synthesized to kill other schistosome species. We can now restructure OXA to make it more efficacious.

    THU-628 TWO SYSTEMS FOR MODULATING BACK-ELECTRON TRANSFER BETWEEN GUANINE RADICALS AND 2-AMINOPURINE IN DUPLEX DNA

    • Priscilla Garcia ;
    • Eric Stemp ;

    THU-628

    TWO SYSTEMS FOR MODULATING BACK-ELECTRON TRANSFER BETWEEN GUANINE RADICALS AND 2-AMINOPURINE IN DUPLEX DNA

    Priscilla Garcia, Eric Stemp.

    Mount St. Mary's University, Los Angeles, Los Angeles, CA.

    Guanine is the DNA base that is most susceptible to 1-electron oxidation, which can lead to mutations. Here, we used the fluorescent analogue 2-aminopurine as a photooxidant of guanine, and examined 2 different DNA systems to determine if the amount of permanent products from guanine oxidation could be modulated. In the first system, we examined DNA-protein crosslinking as the trapping reaction for the guanine radical. We incorporated 2-aminopurine into a 29-mer DNA duplex containing a 5’-G(T)nX-3’ sequence (n = 0 and 4 and X = 2-aminopurine) to determine the relative rates of back-electron transfer and crosslinking to protein. Crosslinking was detected by gel shift assay. Following 325 nm irradiation, little change was seen for the n = 0 duplex, whereas the n = 4 duplex showed the clear appearance of lower mobility bands. This suggests that back-electron transfer does not compete as well with crosslinking in the n = 4 duplex. In the second system, the distance between the photooxidant and the closest guanine was held constant, but the sequence was designed to either increase or decrease in guanine oxidation potential based on the distance from the photooxidant, in order to either localize or push away the electron hole, respectively. Indeed, a less efficient oxidation of the cytochrome c, as evidenced by the loss of the 550 nm absorbance band, was observed for the sequence designed to keep the electron hole in proximity to the photooxidant. Thus, both for DNA-protein crosslinking and for protein oxidation, the extent of reaction with protein can be modulated by design via variations in the DNA sequence.

    FRI-628 EXPRESSION AND PURIFICATION OF HANTAAN AND PUUMALA VIRUS GN GLYCOPROTEINS IN ESCHERICHIA COLI

    • Milagros Hernandez ;
    • Meda Higa ;

    FRI-628

    EXPRESSION AND PURIFICATION OF HANTAAN AND PUUMALA VIRUS GN GLYCOPROTEINS IN ESCHERICHIA COLI

    Milagros Hernandez, Meda Higa.

    York College of Pennsylvania, York, PA.

    Hantaviruses are enveloped, negative-strand-RNA, rodent-borne viruses within the Bunyaviridae family. The genome consists of 3 segments in which one is the M segment. This segment encodes glycoproteins GN and GC that are expressed on the surface of the viral envelope. Both GN and GC hantavirus glycoproteins are critical for cell attachment and entry into host cells. Previous research suggests GC is responsible for membrane fusion in cell entry. However, no information regarding the function of GN or its contribution towards host-cell interactions has been identified. To develop a tool that can better aid researchers in investigating the interactions between viral and host cells, we aimed to produce and purify soluble GN glycoproteins of 2 viruses: HTNV and PUUV. We attempted to express constructs with deleted transmembrane domains in Escherichia coli. Initial results suggested little to no protein production of either HTNV or PUUV soluble GN glycoprotein. Interestingly, however, sequencing results were not inconclusive as they were indicative of the presence of GN glycoprotein in the vector for HTNV. Further research into the optimal conditions for IPTG induction and the solubilization of GN glycoproteins for hantaviruses is being pursued. Soluble GN glycoproteins can be introduced into a chicken for antibody production to identify receptors, mechanisms, and interactions between viral and host cells during cell entry.

    FRI-630 IN VIVO ASSAYS TO DETERMINE RECN MECHANISMS OF ACTION

    • Otto Mossberg ;
    • Shelley Lusetti ;

    FRI-630

    IN VIVO ASSAYS TO DETERMINE RECN MECHANISMS OF ACTION

    Otto Mossberg, Shelley Lusetti.

    New Mexico State University, Las Cruces, NM.

    RecN is an SMC-like bacterial protein associated with recombination. We seek to understand the mechanisms through which RecN assists in recombination to repair DNA double-strand breaks. We constructed strains of E. coli with knockouts for 1, 2, or 3 of the recombination-associated proteins RecN, RecB, RecO, and SbcC in various combinations. Strains are grown in LB liquid media, then a serial dilution of each culture is spotted onto LB agar plates. DNA damage is induced either by exposure to varying doses of UV radiation or by addition of the chemicals ciprofloxacin or azidothimidine to the agar. Colony growth is then assayed to determine genetic interactions between RecN and the other recombination-associated gene products. We hypothesize that RecO, RecB, and RecN constitute 3 epistasis groups and predict the triple mutant is recombination-deficient. Preliminary results show that both the RecN/RecO and RecN/SbcC double mutants have a somewhat reduced capacity for recombination, and the RecN/RecB double mutants have a highly reduced capacity. This suggests that the 3 proteins are involved in separate pathways, and knocking out each protein interferes with 1 of those pathways. Additional assays using the RecN/RecB/RecO triple mutant will allows us to determine whether this is indeed the case.

    THU-618 CHEMICAL CHARACTERIZATION OF PHYTOLITHS WITH RAMAN SPECTROSCOPY: WHAT IS INSIDE

    • Jessica Sanchez ;
    • Eric Potma ;

    THU-618

    CHEMICAL CHARACTERIZATION OF PHYTOLITHS WITH RAMAN SPECTROSCOPY: WHAT IS INSIDE

    Jessica Sanchez1, Eric Potma2.

    1California State University, Fullerton, Fullerton, CA, 2University of California, Irvine, Irvine, CA.

    Phytoliths are silica bodies formed by biomineralization in plants where soluble monosilicic acid is assimilated by the plant and precipitated within the cells and intracellular spaces as amorphous silica. During mineral precipitation, organic matter (OM) can be trapped in the inorganic matrix and, when the plant dies, phytoliths remain in the soil. These can be collected and used for paleoreconstructions based on morphology and isotope content of the trapped OM. The source of the OM in phytoliths has not been well constrained. This study uses Raman spectroscopy to determine the nature of OM in phytoliths obtained from a previous isotope study. These phytoliths were isolated from modern plants, Sorghum bicolor, growing in 6 different experimental conditions. Our purpose is to determine if phytolith carbon changes with different growing conditions. Ideally, differences in OM type and distribution between these treatments could give information about the OM source. Samples of bilobate phytoliths were analyzed in the C-H stretching region (2,700 - 3,200 cm-1) and the fingerprint region (1,300 - 1,800 cm-1) of the vibrational spectrum. Results show that the average spectra for each treatment exhibits consistent, common vibrational bands; however, there are slight differences between the spectra of phytoliths from each treatment. The data suggest the presence of carbohydrates, lignin, and possibly lipids. Moreover, the OM is heterogeneously distributed throughout the phytoliths. Further research will focus on spectral imaging and Raman surveys of silica structures. Information on the nature of OM in phytoliths may aid in determining a mechanism of entrapment.

    THU-630 INVESTIGATION OF THE DNA DAMAGE-INDUCED PROTEIN DIND AND ITS EFFECT ON DNA SYNTHESIS

    • Zachary Romero ;
    • Shelley Lusetti ;

    THU-630

    INVESTIGATION OF THE DNA DAMAGE-INDUCED PROTEIN DIND AND ITS EFFECT ON DNA SYNTHESIS

    Zachary Romero, Shelley Lusetti.

    New Mexico State University, Las Cruces, NM.

    The integrity of DNA is constantly challenged by exogenous and endogenous factors. Many proteins are involved in this constant battle to maintain genomic stability. The protein RecA is one of many proteins up regulated during the bacterial DNA damage response. It serves various functions, one of which is homologous recombination at the site of DNA double-stranded breaks. RecA carries out this function by forming nucleoprotein filaments on the single-stranded regions of DNA, and by mediating strand invasion of another homologous DNA molecule. After DNA pairing, RecA disengages from the DNA, and DNA synthesis occurs. Our lab is interested in another DNA damage-induced protein, DinD, that has been shown to inhibit RecA’s activity. Previous work done in our lab has shown that when DinD is added to a reaction where RecA is performing DNA pairing, DinD stops the reaction. Inhibition of RecA’s function could be attributed to sequestering DNA binding sites from the recombinase. However, further analysis also showed that DinD was not just sequestering binding sites on DNA, but instead it was disassembling the nucleoprotein filament of RecA. The in vivo functions of this activity are not completely understood. However, it is reasonable to think that, under heavy DNA damage, it is important for RecA to disengage from the DNA and allow faster synthesis. Here, we present experiments designed to investigate whether DinD acts this way under DNA stress to allow for efficient DNA synthesis.

    FRI-629 A SMALL COPPER COMPONENT/CARRIER DETECTED IN BLOOD PLASMA THAT BINDS 67CU IMMEDIATELY AFTER UPTAKE

    • Miguel Tellez ;
    • Maria Linder ;

    FRI-629

    A SMALL COPPER COMPONENT/CARRIER DETECTED IN BLOOD PLASMA THAT BINDS 67CU IMMEDIATELY AFTER UPTAKE

    Miguel Tellez, Maria Linder.

    California State University, Fullerton, Fullerton, CA.

    Copper’s biological redox activity is potentially toxic and is thus carefully regulated. Wilson disease can lead to accumulation of excess copper in liver and copper toxicosis and is due to the lack of a functional ATP7B transporter that packs copper into the bile for excretion. Wilson disease model (Atp7b-/-) mice excrete high levels of copper in urine in the form of a 1.5 kDa small copper carrier (SCC). We explored the possibility of the same SCC existing in the blood plasma of mammals. FPLC-SEC data indicate SCC is present and riding on proteins > 200 kDa, from which it is released by EDTA, but SCC is not Cu-EDTA. Large amounts of free SCC are in the blood plasma of Atp7b-/- mice accounting for its occurrence in their urine. SCC accounted for approximately 10% of the total copper in human plasma. To begin to determine how SCC was formed, we treated adult female rats with traces of high-specific-activity radioactive 67Cu by intraperitoneal injection (ip) or gastric intubation, and followed the radioactivity in blood plasma, SCC, and organs over time. High-specific activity 67Cu injected ip into adult, female rats was associated with blood plasma SCC as early as 15 min later. This was also the case when 67Cu was given by gastric intubation, measured as percent dose in organs and plasma/SCC over time. These findings are consistent with the idea that SCC is a part of the albumin and transcuprein exchangeable-copper pool since SCC rapidly binds incoming 67Cu.